Estrogen receptor mutations and uses thereof

ABSTRACT

Novel mutant ESR1 molecules and uses are disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/251,856, now abandoned, which is a continuation of PCT/US2012/060133,filed Oct. 12, 2012, which claims the benefit of U.S. ProvisionalApplication No. 61/547,471, filed Oct. 14, 2011, and U.S. ProvisionalApplication No. 61/615,821, filed Mar. 26, 2012. The contents of eachthese prior applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 14, 2014, isnamed Sequence Listing and is 30,718 bytes in size.

BACKGROUND

Cancer represents the phenotypic end-point of multiple genetic lesionsthat endow cells with a full range of biological properties required fortumorigenesis. Indeed, a hallmark genomic feature of many cancers,including, for example, breast cancer, prostate cancer, ovarian cancer,endometrial cancer, and colon cancer, is the presence of numerouscomplex chromosome structural aberrations, including translocations,intra-chromosomal inversions, point mutations, deletions, gene copynumber changes, gene expression level changes, and germline mutations,among others.

The need still exists for identifying novel genetic lesions associatedwith cancer. Such genetic lesions can be an effective approach todevelop compositions, methods and assays for evaluating and treatingcancer patients.

SUMMARY

The invention is based, at least in part, on the discovery of novelmutations in the Estrogen Receptor 1 (ESR1) gene in a cancer, e.g., abreast, a colorectal, or a lung, cancer. In one embodiment, the mutationis a deletion in the ligand binding domain of the ESR1 gene, e.g., thehuman ESR1 gene. In other embodiments, the mutation is a missensemutation in the hinge domain or the ligand binding domain of the ESR1gene, e.g., the human ESR1 gene.

In one embodiment, a mutant ESR1 (e.g., a mutant ESR1 nucleic acid orpolypeptide) includes a mutation in the hinge domain and/or the ligandbinding domain, e.g., a missense mutation or an in-frame deletion of 3or more nucleotides, in one or more of ESR1 exons, e.g., exons 8-12 ofESR1 (FIG. 1). For example, an ESR1 mutation includes a 6 nucleotidedeletion of nucleotides 1046-1051 (TGGCAG) according to SEQ ID NO:1,which results in the deletion of amino acids 349-351 (LAD) and aninsertion of H at amino acid position 349 (FIG. 4B). In otherembodiments, an ESR1 mutation includes a mutation, e.g., a missensemutation, shown in Table 3, e.g., one or more of mutations at position311, 341, 350, 394, 414, 433, 503, 537 or 538 of the amino acid sequenceof SEQ ID NO:2 (FIGS. 2A-2B); or one or more mutations at positions 932,1022, 1049, 1181, 1240, 1297, 1507, 1609, 1610 or 1613 of the nucleotidesequence of SEQ ID NO:1 (FIGS. 2A-2B). In one embodiment, the ESR1mutation includes a missense mutation at amino acid 538 of SEQ ID NO:2,or nucleotide 1613 of SEQ ID NO:1. In one embodiment, the missensemutation is chosen from one or more of: a threonine to methioninesubstitution at position 311 (a T311M); a serine to leucine substitutionat position 341 (a S341L); an alanine to glutamate substitution atposition 350 (a A350E); an arginine to histidine substitution atposition 394 (a R394H); a glutamine substitution at position 414, e.g.,an insertion to a stop codon (a Q414*); a serine to proline substitutionat position 433 (a S433P); an arginine to tryptophan substitution atposition 503 (a R503W); a tyrosine to asparagine substitution atposition 537 (a Y537N); a tyrosine to cysteine substitution at position537 (a Y537C); or an aspartate to glycine substitution at position 538(a D538G), of SEQ ID NO:2. In one embodiment, the mutation is anaspartate to glycine substitution at position 538 (a D538G), of SEQ IDNO:2. In other embodiments, the mutation is a nucleotide mutation chosenfrom one or more of: a C to T replacement at nucleotide 932; a C to Treplacement at nucleotide 1022; a C to A replacement at nucleotide 1049;a G to A replacement at nucleotide 1181A; a C to T replacement atnucleotide 1240; a T to C replacement at nucleotide 1297; a T to Areplacement at nucleotide 1609; an A to G replacement at nucleotide1610; or an A to G replacement at nucleotide 1613, of SEQ ID NO:1. Inone embodiment, the mutation is an A to G replacement at nucleotide1613, of SEQ ID NO:1. In one embodiment, the mutation is an insertion ofa C between amino acids G344 and L345 of SEQ ID NO:2; an insertion ofnucleotides GCT between nucleotides 1032 and 1033 of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the hingeregion, e.g., a mutation that causes an amino acid substitution atposition 311 of SEQ ID NO:2, e.g., a T311M mutation. In one embodiment,the mutation features a C932T substitution according to the sequence ofSEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid substitutionat position 341 of SEQ ID NO:2, e.g., a S341L mutation. In oneembodiment, the mutation features a C1022T substitution according to thesequence of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid insertionbetween amino acids G344 and L345 of SEQ ID NO:2, e.g., an insertion ofa C amino acid. In one embodiment, the mutation features an insertion ofnucleotides GCT between nucleotides 1032 and 1033 of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid substitutionat position 350 of SEQ ID NO:2, e.g., an A350E mutation. In oneembodiment, the mutation features a C1049A substitution according to thesequence of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid substitutionat position 394 of SEQ ID NO:2, e.g., an R394H mutation. In oneembodiment, the mutation features a G1181A substitution according to thesequence of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that results in a truncated proteinthat ends at position 413 of SEQ ID NO:2, e.g., a Q414* mutation, due toa mutation in the nucleotide sequence that introduces a stop codon,e.g., a C1240T substitution according to the sequence of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid substitutionat position 433 of SEQ ID NO:2, e.g., an S433P mutation. In oneembodiment, the mutation features a T1297C substitution according to thesequence of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid substitutionat position 503 of SEQ ID NO:2, e.g., an R503W mutation. In oneembodiment, the mutation features a C1507T substitution according to thesequence of SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid substitutionat position 537 of SEQ ID NO:2, e.g., an Y537N mutation or a Y537C. Inone embodiment, the mutation features a Y537N mutation according to thesequence of SEQ ID NO:2, and a corresponding T1609A substitutionaccording to the sequence of SEQ ID NO:1. In another embodiment, themutation features a Y537C mutation according to the sequence of SEQ IDNO:2, and a corresponding A1610G substitution according to the sequenceof SEQ ID NO:1.

In one embodiment, a mutant ESR1 includes a mutation in the ligandbinding domain, e.g., a mutation that causes an amino acid substitutionat position 538 of SEQ ID NO:2, e.g., an D538G mutation. In oneembodiment, the mutation features a A1613G substitution according to thesequence of SEQ ID NO:1.

A mutant polypeptide encoded by a mutant ESR1 nucleic acid is sometimesreferred to herein as a “mutant ESR1 polypeptide.” In an embodiment, themutant ESR1 polypeptide can have an alteration in one or more of thefollowing activities: a ligand binding activity (e.g., an alteration inits ability to bind to estrogen or an estrogen mimetic), a DNA bindingactivity, an interaction with a co-repressor, an interaction with aco-activator (e.g., to activate transcription). In one embodiment, themutant ESR1 polypeptide has an altered ligand binding activity, analtered dimerization activity, an altered translocation activity, analtered AF-2 function, and/or impaired ER signaling, e.g., compared toan ESR1 polypeptide that does not have the mutation, e.g., a wild typeESR1 polypeptide. In other embodiments, the mutant ESR1 polypeptide isconstitutively active, e.g., has a constitutively active transcriptionalactivity, compared to an ESR1 polypeptide that does not have themutation, e.g., a wild type ESR1 polypeptide. In certain embodiments,the mutant ESR1 polypeptide has at least one activity that is notsubstantially affected by an ESR1 agonist or antagonist (e.g.,estradiol, tamoxifen or other anti-estrogen agent or “Selective EstrogenReceptor Modulator” (SERM)). In other embodiment, the mutant ESR1polypeptide has altered (e.g., reduced) phosphorylation at position 537of SEQ ID NO:2. In some embodiments, a mutant ESR1 is activated, or hasenhanced activity when contacted with a SERM, such as tamoxifen, ascompared to the activity of an ESR1 polypeptide that does not carry themutation. An enhanced activity of a mutant ESR1 can exhibit constitutiveor enhanced activity, e.g., in a cell of a cancer referred to herein(e.g., a breast cancer (e.g., a SERM-resistant breast cancer), acolorectal cancer, or a lung cancer). The constitutive or enhancedactivity can be enhanced transcriptional activation as compared to anESR1 that does not carry the mutation. A cell of a cancer can carry anESR1 nucleic acid and polypeptide that contains a mutation in the ligandbinding domain described herein, e.g., a 6 nucleotide deletion in theligand binding domain, an insertion or missense mutation, and can alsocarry an ESR1 nucleic acid and polypeptide that does not carry themutation.

In one embodiment, the mutant ESR1 comprises at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12 exons from ESR1. In another embodiment, the mutantESR1 polypeptide includes an activation function domain-1 (AF-1), a DNAbinding domain, a hinge domain, and a ligand binding domain containingan activation function 2 (AF-2) domain, or a functional fragmentthereof. In another embodiment, the mutant ESR1 polypeptide includes aDNA binding domain, a hinge domain, and a ligand binding domaincontaining an activation function 2 (AF-2) domain, or a functionalfragment thereof.

In certain embodiments, the mutant ESR1 is identified in a subject,e.g., a patient who is positive for an estrogen receptor (e.g., an ERpositive (ER+) subject as detected, e.g., by immunohistochemistry). Inother embodiments, the subject is identified, or has been previouslyidentified, as having a cancer with an ER+ status (e.g., a cancer thatgrows in response to an estrogen). In certain embodiments, the subjecthas a localized cancer, e.g., a localized breast cancer. In otherembodiments, the subject has a metastatic cancer. In certain embodiment,the subject has a late stage or advanced breast cancer, e.g., a latestate progressive metastatic cancer. In other embodiments, the subjecthas an ER positive, late stage or advance breast cancer, e.g., an ERpositive, metastatic breast cancer. In one embodiment, the subject is anER+ breast cancer patient with late stage, progressive metastaticcancer.

In certain embodiments, the subject is currently undergoing treatmentwith a SERM (e.g., tamoxifen) or other anti-estrogen. In otherembodiments, the subject is currently undergoing treatment with anaromatase inhibitor. In yet other embodiments, the subject is undergoingtreatment with chemotherapy. In other embodiment, the subject isundergoing treatment with a Her 2 inhibitor, e.g., an anti-Her 2antibody (e.g., HERCEPTIN). In certain embodiments, the subject (e.g.,an ER+ subject) shows a decrease response or is non-responsive (orresistant) to a treatment with one or more of SERM, an aromataseinhibitor, a chemotherapy, or a Her 2 inhibitor. In certain embodiment,the subject is further evaluated for the presence of one or more of themutations disclosed herein. In one embodiment, the treatment of thesubject, e.g., the treatment with one or more of the SERM, the aromataseinhibitor, the chemotherapy, or the Her 2 inhibitor is modified, e.g.,decreased, discontinued, or otherwise altered, in response to thedetection of one or more of the mutations described herein. In oneembodiment, the treatment modified is a treatment with the SERM.

In one embodiment, identification of a mutation in the ligand bindingdomain of ESR1 is indicative that the cancer is less responsive tohormone therapy (e.g., the cancer is hormone-resistant). In certainembodiment, the presence of an ESR mutant is indicative of diseaseprogression. In certain embodiments, the mutation is not identified inthe primary cancer. In other embodiments, the mutation is identified inmetastatic cancer. In certain embodiment, the mutation is identified ina late stage or advanced breast cancer, e.g., a late state progressivemetastatic cancer. In other embodiments, the mutation is identified inan ER positive, late stage or advance breast cancer, e.g., an ERpositive, metastatic breast cancer. In one embodiment, the mutation isidentified in an ER+ breast cancer patient with late stage, progressivemetastatic cancer.

In certain embodiments, a cancer patient having a mutation in the ligandbinding domain of ESR1 can be administered an anti-cancer therapy thatis not a SERM. In one embodiment, the other anti-cancer therapy is anaromatase inhibitor. In other embodiments, the other therapy is an mTOR(mammalian Target of Rapamycin) pathway inhibitor, for example, RAD001(everolimus), CCI-799 (tensirolimus), and AP23573 (ARIAD). For example,in one embodiment, the subject is a post-menopausal female, andidentification of a mutation in the ligand binding domain of ESR1,indicates that a cancer patient can be administered an estrogeninhibitor, such as an aromatase inhibitor or fulvestrant, alone or incombination with an mTOR pathway inhibitor. In another embodiment, thesubject is a premenopausal female, and the identification of a mutationin the ligand binding domain of ESR1 indicates that the patient can beadministered an estrogen receptor blocking agent or should have anoophorectomy (ovary removal). In one embodiment, identification of amutation in the ligand binding domain of ESR1 indicates that a cancerpatient should not be administered a SERM, such as tamoxifen orraloxifene. In yet other embodiments, a post-menopausal female or apremenopausal female identified as carrying a mutation in the ligandbinding domain of ESR continues to receive treatment with a SERM (e.g.,at the same or a reduced dose of the SERM). In other embodiments, theanti-cancer therapy includes an aromatase inhibitor (e.g., examestane),alone or in combination with an mTOR pathway inhibitor, for example,RAD001 (everolimus).

In one embodiment, a subject, e.g., a cancer patient is alreadyreceiving therapy with a SERM, e.g., tamoxifen, and the identificationof a mutation in ESR1 indicates that the patient can receive analternate therapy, e.g., the patient should stop receiving treatmentwith a SERM, or reduce the dose of a SERM, and/or begin therapy with analternative therapy.

In one embodiment, the alternative therapy includes an aromataseinhibitor (e.g., anastrozole), a Selective Estrogen ReceptorDownregulator (SERD) or anti-estrogen (e.g., fulvestrant), or an mTORpathway inhibitor, or a combination thereof.

In another embodiment, the alternate therapy reduces or inhibits theconstitutive activation of an ESR1, e.g., a mutant ESR1 as describedherein. In one embodiment, the alternative therapy reduces or inhibitsone or more of: a ligand binding activity, receptor translocation, DNAbinding activity, receptor dimerization, or an interaction with aco-activator (e.g., to activate transcription). In other embodiments,the alternative therapy reduces the level of an ESR1, e.g., a mutantESR1 as described herein (e.g., a constitutively active ESR1). Forexample, the alternative therapy reduces the expression or the stabilityof an ESR1 gene product; and/or increases ESR1 degradation. In oneembodiment, the alternative therapy includes administration a SelectiveEstrogen Receptor Downregulator (SERD). In one embodiment, thealternative therapy is a non-steroidal ERα antagonist that induces ERαdegradation. In other embodiments, the alternative therapy inhibits orreduces ERα transcriptional activity and/or steady state levels.Exemplary estrogen receptor modulators that can be used as analternative therapy are described in, e.g., WO 2012/037411, WO2012/037410, WO 2011/159769, WO 2011/156518 and US 2012/0071535, thecontents of all of which are hereby incorporated by reference. Forexample, exemplary estrogen receptor modulators described in WO2012/037411 include compounds presented in Table 5, or apharmaceutically acceptable salt thereof:

TABLE 5 Exemplary estrogen receptor modulators described in WO2012/037411 Com- LCMS pound Name Structure [M + H]⁺ 1 (E)-Ethyl3-(4-((E)-1-(1H-indazol-5-yl)-2- phenylbut-1-en-1-yl)phenyl)acrylate

423 2 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

395 3 (E)-3-(4-((E)-1-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

396 4 (E)-3-(4-((E)-2-(4-Fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

413 5 (E)-3-(4-((E)-1-(1H-Indazol-6-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

395 6 (E)-3-(4-((E)-2-(4-Chlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

429 7 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-methoxyphenyl)but-1-en-1-yl)phenyl) acrylic acid

425 8 (E)-3-(4-((E)-2-(3-(Hydroxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

425 9 (E)-3-(4-((E)-2-(4-(Hydroxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

425 10 (E)-3-(4-((E)-2-(2-(Hydroxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

425 11 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(o-tolyl)but-1-en-1-yl)phenyl)acrylic acid

409 12 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(m-tolyl)but-1-en-1-yl)phenyl)acrylic acid

409 13 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(p-tolyl)but-1-en-1-yl)phenyl)acrylic acid

409 14 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methoxyphenyl)but-1-en-1-yl)phenyl) acrylic acid

425 15 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-methoxyphenyl)but-1-en-1-yl)phenyl) acrylic acid

425 16 ((E)-3-(4-((E)-2-(2-Chlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

429 17 (E)-3-(4-((E)-2-(3-Chlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

429 18 (E)-3-(4-((E)-1-(1H-Indazol-4-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

395 19 (E)-3-(4-((E)-2-(2-Fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

413 20 (E)-3-(4-((E)-2-(3-Fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

413 21 (E)-3-(4-((E)-1-(1H-Indazol-4-yl)-2-(o-tolyl)but-1-en-1-yl)phenyl)acrylic acid

409 22 (E)-3-(4-((E)-2-(2-Chlorophenyl)-1-(1H-indazol-4-yl)but-1-en-1-yl)phenyl)acrylic acid

429 23 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(thiophen-2-yl)but-1-en-1-yl)phenyl)acrylic acid

401 24 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(thiophen-3-yl)but-1-en-1-yl)phenyl)acrylic acid

401 25 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(pyridin-2-yl)but-1-en-1-yl)phenyl)acrylic acid

396 26 (E)-3-(4-((E)-2-(2-Ethylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

423 27 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-(trifluoromethyl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

463 28 (E)-3-(4-((E)-2-([1,1′-Biphenyl]-2-yl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl) acrylic acid

471 29 (E)-3-(4-((E)-4-Chloro-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

429 30 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-4-yl)but-1-en-1- yl)phenyl)acrylic acid

447 31 (E)-3-(4-((E)-2-(2-Fluorophenyl)-1-(1H-indazol-4-yl)but-1-en-1-yl)phenyl)acrylic acid

413 32 (E)-3-(4-((E)-2-(2-Cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

420 33 (E)-3-(4-((E)-2-(2,4-Difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl) acrylic acid

431 34 (E)-3-(4-((E)-2-(2-Chloro-3-fluorophenyl)-1-(1H-indaozl-5-yl)but-1-en-1- yl)phenyl)acrylic acid

447 35 (E)-3-(4-((E)-2-Cyclopropyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl) acrylic acid

407 36 (E)-3-(4-((E)-2-(4-Fluoro-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

427 37 (E)-3-(4-((E)-2-(2,6-Difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl) acrylic acid

431 38 (E)-3-(4-((E)-2-(2,6-Dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl) acrylic acid

463 39 (E)-3-(4-((E)-4,4,4-Trideutero-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

398 40 (E)-3-(4-((E)-2-(4-Fluoro-3-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

427 41 (E)-3-(4-((E)-2-(5-Fluoro-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

427 42 (E)-3-(4-((E)-2-(2,3-Difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl) acrylic acid

431 43 (E)-3-(4-((E)-2-(2,5-Difluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl) acrylic acid

431 44 (E)-3-(4-((E)-2-(2-Chloro-5-luorophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

447 45 (E)-3-(4-((E)-2-(2-Chloro-6-methylphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

443 46 (E)-3-(4-((E)-1-(7-Chloro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

429 47 (E)-3-(4-((E)-1-(4-Methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

409 48 (E)-3-(4-((E)-1-(7-Methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

409 49 (E)-3-(4-((E)-1-(6-Methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

409 50 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2- (naphthalen-1-yl)but-1-en-1-yl)phenyl)acrylic acid

445 51 (E)-3-(4-((E)-1-(3-Methyl-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

409 52 (E)-3-(4-((E)-1-(3-Chloro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

429 53 (E)-3-(4-((E)-2-(4-Chloro-2-methylphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

443 54 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylprop-1-en-1-yl)phenyl)acrylic acid

381 55 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylpent-1-en-1-yl)phenyl)acrylic acid

409 56 (E)-3-(4-((E)-2-Phenyl-1-(1H-pyrazolo[3,4-b]pyridin-4-yl)but-1-en-1-yl)phenyl) acrylic acid

396 57 (E)-3-(4-((E)-2-(3-Cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

420 58 (E)-3-(4-((E)-2-(4-Cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

420 59 (E)-3-(4-((E)-4-Hydroxy-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

411 60 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-4- methoxy-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

425 61 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-3- methoxy-2-phenylprop-1-en-1-yl)phenyl)acrylic acid

411 62 (E)-3-(4-((E)-2-(4-(Dimethylamino)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

438 63 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

413 64 (E)-3-(4-((E)-1-(1H-Indol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

394 65 (E)-3-(4-((E)-1-(6-Chloro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

429 66 (E)-3-(4-((E)-1-(2-Oxo-2,3-dihydrobenzo[d]oxazol-6-yl)-2-phenylbut- 1-en-1-yl)phenyl)acrylic acid

412 67 (E)-3-(4-((E)-2-Phenyl-1-(1H-pyrazolo[3,4-b]pyridin-5-yl)but-1-en-1-yl)phenyl) acrylic acid

396 68 (E)-3-(4-((E)-2-Phenyl-1-(1H-pyrrolo[2,3-b]pyridin-5-yl)but-1-en-1-yl)phenyl)acrylic acid

395 69 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-4-methyl-2-phenylpent-1-en-1-yl)phenyl) acrylic acid

423 70 (E)-3-(4-((E)-2-Cyclopropyl-1-(1H-indazol-4-yl)-2-phenylvinyl)phenyl)acrylic acid

407 71 (E)-3-(4-((E)-2-(2-Chlorophenyl)-2- cyclopropyl-1-(1H-indazol-4-yl)vinyl)phenyl)acrylic acid

441 72 (E)-3-(4-((E)-1-(1H-Benzo[d][1,2,3]triazol-5-yl)-2-(2-chloro-4-fluorophenyl) but-1-en-1-yl)phenyl)acrylicacid

448 73 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-2-cyclopropyl-1-(1H-indazol-4- yl)vinyl)phenyl)acrylic acid

459 74 (E)-3-(4-((E)-1-(4-Chloro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

429 75 (E)-3-(4-((E)-1-(2-Oxo-2,3-dihydrobenzo[d]thiazol-6-yl)-2-phenylbut- 1-en-1- yl)phenyl)acrylic acid

428 76 (E)-3-(4-((E)-2-Cyclopentyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid

435 77 (E)-3-(4-((E)-2-Cyclohexyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid

449 78 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-3-methyl-2-phenylbut-1-en-1-yl)phenyl) acrylic acid

409 79 (E)-3-(4-((E)-3-Cyclopropyl-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl) phenyl)acrylic acid

421 80 (E)-3-(4-((E)-2-(2-Chlorophenyl)-2- cyclopropyl-1-(1H-indazol-5-yl)vinyl)phenyl)acrylic acid

441 81 (E)-3-(4-((E)-2-Phenyl-1-(1H-pyrazolo[3,4-c]pyridin-5-yl)but-1-en-1-yl)phenyl) acrylic acid

396 82 (E)-3-(4-((E)-1-(6-Fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

413 83 (E)-3-(4-((E)-1-(1H-Benzo[d]imidazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

395 84 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylhex-1-en-1-yl)phenyl)acrylic acid

423 85 (E)-3-(4-((E)-3-Cyclopentyl-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl) phenyl)acrylic acid

449 86 (E)-3-(4-((E)-1-(6-Fluoro-1H-indazol-4-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

413 87 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

465 88 (E)-3-(4-((E)-1-(1H-Benzo[d][1,2,3]triazol-4-yl)-2-phenylbut-1-en-1-yl)phenyl) acrylic acid

396 89 (E)-3-(4-((E)-1-(1H-Benzo[d][1,2,3]triazol-4-yl)-2-(2-chloro-4-fluorophenyl)but-1-en- 1-yl)phenyl)acrylic acid

448 90 (E)-3-(4-((E)-1-(3-Oxo-2,3-dihydroisoquinolin-6-yl)-2-phenylbut-1- en-1-yl)phenyl)acrylic acid

422 91 (E)-3-(4-((E)-1-(7-Fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

413 92 (E)-3-(4-((E)-1-(7-Fluoro-1H-indol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

412 93 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)-4-methylpent-1-en-1- yl)phenyl)acrylic acid

475 94 (E)-3-(4-((Z)-3,3-Difluoro-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl)phenyl) acrylic acid

417 95 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(7-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

465 96 (E)-3-(4-((E)-2-(Benzofuran-4-yl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

435 97 (E)-3-(4-((E)-1-(1-Oxo-1,2-dihydroisoquinolin-6-yl)-2-phenylbut-1- en-1-yl)phenyl)acrylic acid

422 98 (E)-3-(4-((E)-4-Fluoro-1-(1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

413 99 (E)-3-(4-((E)-4-Chloro-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

481 100 (E)-3-(4-((E)-1-(2-Oxo-1,2,3,4-tetrahydroquinolin-6-yl)-2-phenylbut-1-en- 1-yl)phenyl)acrylic acid

424 101 (E)-3-(4-((E)-1-(2-Oxo-2,4-dihydro-1H-benzo[d][1,3]oxazin-6-yl)-2-phenylbut-1- en-1-yl)phenyl)acrylic acid

426 102 (E)-3-(4-((E)-1-(2-Oxo-1,2- dihydroquinolin-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

422 103 (E)-3-(4-((E)-1-(3-Oxo-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)-2-phenylbut-1- en-1-yl)phenyl)acrylic acid

442 104 (E)-3-(4-((E)-1-(3-Oxoisoindolin-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

410 105 (E)-3-(4-((E)-1-(2-Oxo-1,2-dihydroquinolin-6-yl)-2-phenylbut-1-en- 1-yl)phenyl)acrylic acid

422 106 (E)-3-(4-((Z)-3,3,3-Trifluoro-1-(1H-indazol-5-yl)-2-phenylprop-1-en-1-yl) phenyl)acrylic acid

Not Observed 107 (E)-3-(4-((E)-1-(3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-2-phenylbut- 1-en-1-yl)phenyl)acrylic acid

Not Observed 108 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(4-fluoro-2-methylphenyl)but-1- en-1-yl)phenyl)acrylic acid

445 109 (E)-3-(4-((E)-2-(4-Chloro-2-methylphenyl)-1-(4-fluoro-1H-indazol-5- yl)but-1-en-1-yl)phenyl)acrylicacid

461 110 (E)-3-(4-((E)-1-(1-Oxoisoindolin-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

410 111 (E)-3-(4-((E)-2-Cyclopropyl-1-(4-fluoro-1H-indazol-5-yl)-2-(4-fluoro-2- methylphenyl)vinyl)phenyl)acrylic acid

457 112 (E)-3-(4-((E)-2-(4-Chloro-2-methylphenyl)-2-cyclopropyl-1-(4-fluoro-1H-indazol-5-yl)vinyl)phenyl)acrylic acid

473 113 (E)-3-(4-((E)-1-(4-Chloro-1H-indazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1- en-1-yl)phenyl)acrylic acid

481 114 (E)-3-(4-((Z)-2-(2-Chloro-4- fluorophenyl)-3,3-difluoro-1-(1H-indazol-5-yl) prop-1-en-1-yl)phenyl)acrylic acid

469 115 (E)-3-(4-((E)-2-Cyclopropyl-1-(4-fluoro-1H-indazol-5-yl)-2-phenylvinyl) phenyl)acrylic acid

425 116 (E)-3-(4-((E)-4-Chloro-1-(4-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

447 117 (E)-3-(4-((E)-4-Chloro-2-(2-chloro-4-fluorophenyl)-1-(4-fluoro-1H-indazol-5- yl)but-1-en-1-yl)phenyl)acrylicacid

499 118 (E)-3-(4-((E)-2-(4-Fluorophenyl)-1-(quinolin-6-yl)but-1-en-1-yl)phenyl) acrylic acid

406 119 (E)-3-(4-((E)-4-fluoro-2-(4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

431 120 (E)-3-(4-((E)-2-Phenyl-1-(1H-pyrazolo[4,3-b]pyridin-5-yl)but-1-en-1-yl)phenyl) acrylic acid

396 121 (E)-3-(4-((E)-4-Fluoro-1-(4-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

431 122 (E)-3-(4-((E)-1-(2-Oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2-phenylbut-1- en-1-yl)phenyl)acrylic acid

411 123 (E)-3-(4-((E)-1-(Naphthalen-2-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

404 124 (E)-3-(4-((E)-2-Phenyl-1-(quinoxalin-6-yl)but-1-en-1-yl)phenyl)acrylic acid

407 125 (E)-3-(4-((E)-2-Phenyl-1-(pyrazolo[1,5-a]pyridin-5-yl)but-1-en-1-yl)phenyl) acrylic acid

395 126 (E)-3-(4-((E)-1-(Imidazo[1,2-a]pyridin-6-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

395 127 (E)-3-(4-((E)-1-([1,2,4]Triazolo[4,3-a]pyridin-6-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

396 128 (E)-3-(4-((E)-1-(Benzo[d][1,3] dioxol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

397 (ES−) 129 (E)-3-(4-((E)-1-(Imidazo[1,2-a]pyridin-6-yl)-2-(o-tolyl)but-1-en-1-yl) phenyl)acrylic acid

409 130 (E)-3-(4-((E)-1-(Benzo[d]thiazol-6-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

412 131 (E)-3-(4-((E)-1-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

396 132 (E)-3-(4-((E)-2-Phenyl-1-(tetrazolo[1,5-a]pyridin-6-yl)but-1-en-1-yl)phenyl) acrylic acid

397 133 (E)-3-(4-((E)-1-(Imidazo[1,5-a]pyridin-6-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

395 134 (E)-3-(4-((E)-1-([1,2,4]Triazolo[4,3-a]pyridin-7-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

396 135 (E)-3-(4-((E)-1-(Imidazo[1,5-a]pyridin-7-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

395 136 (E)-3-(4-((E)-1-([1,2,4]Triazolo[1,5-a]pyridin-7-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

396 137 (E)-3-(4-((E)-1-(Benzo[c][1,2,5]oxadiazol-5-yl)-2-phenylbut-1-en-1-yl) phenyl)acrylic acid

397 138 (E)-3-(4-((E)-1-([1,2,3]Triazolo[1,5-a]pyridin-5-yl)-2-phenylbut-1-en-1- yl)phenyl)acrylic acid

396 139 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-5-methoxy-2-phenylpent-1-en-1- yl)phenyl)acrylic acid

439 140 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-6- methoxy-2-phenylhex-1-en-1-yl)phenyl)acrylic acid

453 141 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

412 142 (E)-3-(4-((E)-1-(Benzo[b]thiophen-2-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

409 143 (E)-3-(4-((E)-2-Phenyl-1-(1-phenyl-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

471 144 (E)-3-(4-((E)-1-(Benzo[c][1,2,5]thiadiazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl) acrylic acid

413 145 (E)-3-(4-((E)-2-Phenyl-1-(1-(pyridin-3-yl)-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

472 146 (E)-3-(4-((E)-1-(Benzo[d]oxazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

396 147 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-phenoxyphenyl)but-1-en-1-yl)phenyl) acrylic acid

487 148 (E)-3-(4-((E)-2-Phenyl-1-(pyrazolo[1,5-a]pyridin-3-yl)but-1-en-1-yl)phenyl) acrylic acid

395 149 (E)-3-(4-((E)-2-Phenyl-1-(1H-pyrazolo[4,3-d]pyrimidin-5-yl)but-1-en-1- yl)phenyl)acrylic acid

397 150 (E)-3-(4-((E)-2-(3- (Carboxymethoxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

345 151 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)-3-methylbut- 1-en-1-yl)phenyl)acrylic acid

461 152 (E)-3-(4-((E)-1-(6-Hydroxypyridin-3-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

372 153 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(trifluoromethoxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

479 154 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-3-methyl-2-(4-methylthiophen-3-yl) but-1-en-1-yl)phenyl)acrylic acid

429 155 (E)-3-(4-((E)-2-Cyclobutyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid

421 156 (E)-3-(4-((E)-1-(Benzo[d]isothiazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

412 157 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-2-cyclobutyl-1-(1H-indazol-5- yl)vinyl)phenyl)acrylic acid

473 158 (E)-3-(4-((E)-1-(3-Fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl) acrylic acid

413 159 (E)-3-(4-((E)-1-(2-Methylbenzo[d]thiazol-5-yl)-2-phenylbut-1-en-1-yl) phenyl)acrylic acid

426 160 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(4-(pyrimidin-2-yloxy)phenyl) but-1-en-1-yl)phenyl)acrylic acid

507 161 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(4-(pyrazin-2-yloxy)phenyl) but-1-en-1-yl)phenyl)acrylic acid

507 162 (E)-3-(4-((E)-2-Cyclobutyl-1-(1H-indazol-5-yl)-2-(4-((5-(methylsulfonyl)pyridin-2-yl)oxy)phenyl)vinyl)phenyl)acrylic acid

592 163 (E)-3-(4-((E)-1-(7-Methoxybenzofuran-3-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

Not Observed 164 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(7-methoxybenzofuran-3-yl)but-1-en-1- yl)phenyl)acrylic acid

477 165 (E)-3-(4-((E)-2-Cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)-2-phenylvinyl) phenyl)acrylic acid

439 166 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(3-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

465 167 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-2-cyclobutyl-1-(3-fluoro-1H-indazol-5- yl)vinyl)phenyl)acrylic acid

491 168 (E)-3-(4-((E)-1-(Isoquinolin-7-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

405 169 (E)-3-(4-((E)-1-(Benzofuran-2-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-1- yl)phenyl)acrylic acid

447 170 (E)-3-(4-((E)-1-(Benzofuran-3-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-1- yl)phenyl)acrylic acid

447 171 (E)-Ethyl 3-(4-((E)-2-(4-fluroophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylate

441 172 (E)-Ethyl 3-(4-((E)-1-(1H-indazol-6-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylate

423 173 (E)-Ethyl 3-(4-((E)1-(1H-Indazol-5-yl)-2-(2-methoxyphenyl)but-1-en-1- yl)phenyl)acrylate

453 174 (E)-Ethyl 3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-methoxyphenyl)but-1-en-1- yl)phenyl)acrylate

453 175 (E)-Ethyl 3-(4-((E)-1-(1H-Indazol-4-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylate

423 176 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-2- methylacrylic acid

409 177 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2- phenylbut-1-en-1-yl)-3-methylphenyl)acrylic acid

409 178 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2- phenylbut-1-en-1-yl)-2-methylphenyl)acrylic acid

409 179 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)-2-chlorophenyl) acrylic acid

429 180 (E)-3-(6-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)naphthalen-2-yl) acrylic acid

445 181 (Z)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-2- fluoroacrylic acid

413 182 (Z)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-2- chloroacrylic acid

429 183 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)-3-fluorophenyl) acrylic acid

413 184 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)-2-fluorophenyl) acrylic acid

413 185 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2- phenylbut-1-en-1-yl)-2-(trifluoromethyl)phenyl)acrylic acid

463 186 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)thiophen-2-yl)acrylic acid

401 187 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2- phenylbut-1-en-1-yl)-3-methoxyphenyl)acrylic acid

425 188 (E)-3-(4-((Z)-1-(1H-Indazol-5-yl)-2- phenylbut-1-en-1-yl)-2-methoxyphenyl)acrylic acid

425 189 (E)-3-(5-((Z)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)acrylic acid

396 190 (E)-2-(4-(1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenoxy)acetic acid

399 191 (E)-5-(2-Phenyl-1-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)but-1-en-1-yl)-1H- indazole

438 192 (E)-5-(1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)-2,3-dihydro-1H-indene-2- carboxylic acid

409 193 (E)-Ethyl 3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylate

475 [(M − THP + H) + H]+ 194 (E)-Ethyl 3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylatehydrochloride

475 195 (E)-3-(4-((E)-2-(2-Chloro-4- fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

447 196 (E)-Ethyl 3-(4-((E)-2-(2,4- dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylate

491 197 (E)-3-(4-((E)-2-(2,4-Dichlorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl) phenyl)acrylic acid

463 198 (E)-3-(4-((E)-2-(4-Chloro-2-(trifluoromethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

497 199 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-2-cyclopropyl-1-(1H-indazol-5- yl)vinyl)phenyl)acrylic acid

459 200 (E)-3-(4-((E)-2-(4-Fluoro-2-(trifluoromethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

481 201 (E)-3-(4-(1-(4-Fluoro-1H-indazol-5-yl)-2-(4-fluoro-2-(trifluoromethyl) phenyl)butyl)phenyl)acrylic acid

499 202 (E)-3-(4-((E)-2-(2,4-Dichlorophenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

481 203 (E)-3-(4-((E)-2-(4-Chloro-2-(trifluoromethyl)phenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

515 204 (E)-3-(4-((E)-2-(3-Chloropyridin-4-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

430 205 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-4-fluoro-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

465 206 (E)-3-(4-((E)-2-(2-Chloro-4-methoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

459 207 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(trifluoromethyl)pyridin-3-yl)but-1-en-1- yl)phenyl)acrylic acid

464 208 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(1-methyl-1H-pyrazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

399 209 (E)-3-(4-((E)-2-(2,4-Dichlorophenyl)-4-fluoro-1-(4-fluoro-1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

499 210 (E)-3-(4-((E)-2-Cyclopropyl-2-(2,4-dichlorophenyl)-1-(4-fluoro-1H-indazol-5- yl)vinyl)phenyl)acrylic acid

493 211 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-2-cyclopropyl-1-(4-fluoro-1H-indazol-5- yl)vinyl)phenyl)acrylic acid

477 212 (E)-3-(4-((E)-2-(4-(Dimethylcarbamoyl)-2-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en- 1-yl)phenyl)acrylic acid

484 213 (E)-3-(4-((E)-2-(2-Fluoro-4-(methylcarbamoyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

470 214 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methylthiophen-3-yl)but-1-en-1- yl)phenyl)acrylic acid

415 215 (E)-3-(4-((E)-2-Cyclopropyl-2-(2,4-dichlorophenyl)-1-(1H-indazol-5- yl)vinyl)phenyl)acrylic acid

475 216 (E)-3-(4-((E)-2-(4-Chloro-2- methylphenyl)-2-cyclopropyl-1-(1H-indazol-5- yl)vinyl)phenyl)acrylic acid

455 217 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methylpyridin-3-yl)but-1-en-1- yl)phenyl)acrylic acid

410 218 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-methylpyridin-3-yl)but-1-en-1- yl)phenyl)acrylic acid

410 219 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(pyrazin-2-yl)but-1-en-1-yl)phenyl)acrylic acid

397 220 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(pyrimidin-5-yl)but-1-en-1-yl)phenyl) acrylic acid

397 221 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(1-methyl-1H-1,2,3-triazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

400 222 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-(trifluoromethyl)pyridin-3-yl)but-1-en-1- yl)phenyl)acrylic acid

464 223 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-methylthiophen-3-yl)but-1-en-1- yl)phenyl)acrylic acid

415 224 (E)-3-(4-((E)-2-(3-Chloropyridin-4-yl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

448 225 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(isothiazol-4-yl)but-1-en-1-yl)phenyl)acrylic acid

402 226 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-methylpyridin-4-yl)but-1-en-1- yl)phenyl)acrylic acid

410 227 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(5-methylthiophen-2-yl)but-1-en-1- yl)phenyl)acrylic acid

415 228 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methyl-5-(methylsulfonyl)phenyl)but-1-en- 1-yl)phenyl)acrylic acid

487 229 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methylthiazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

416 230 (E)-3-(4-((E)-2-(3-Chloropyridin-4-yl)-4-fluoro-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

448 231 (E)-3-(4-((E)-2-(3-Fluoropyridin-4-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

414 232 (E)-3-(4-((E)-2-(2,4-Dichlorophenyl)-1-(imidazo[1,2-a]pyridin-6-yl)but-1-en-1- yl)phenyl)acrylic acid

463 233 (E)-3-(4-((E)-2-(4-Fluoro-2-(trifluoromethyl)phenyl)-1-(imidazo[1,2-a]pyridin-6-yl)but-1-en-1-yl)phenyl)acrylic acid

481 234 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-methoxy-2-methylphenyl)but-1-en-1- yl)phenyl)acrylic acid

439 235 (E)-3-(4-((E)-2-(2-Fluoro-4-methoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

443 236 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methoxypyridin-4-yl)but-1-en-1- yl)phenyl)acrylic acid

426 237 (E)-3-(4-((E)-2-(2-Chloro-5-methoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

459 238 (E)-3-(4-((E)-2-(2-Fluoro-4-(methylsulfonyl)phenyl)-1-(1H-indazol-5- yl)but-1-en-1-yl)phenyl)acrylicacid

491 239 (E)-3-(4-((E)-2-(2,4-Dichlorophenyl)-3,3,4,4,4-pentadeutero-1-(1H-indazol-5- yl)but-1-en-1-yl)phenyl)acrylicacid

468 240 (E)-3-(4-((E)-2-(Benzo[d][1,3]dioxol-5-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

439 241 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-methylthiophen-2-yl)but-1-en-1- yl)phenyl)acrylic acid

415 242 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(4-methylthiophen-3-yl)but-1-en-1- yl)phenyl)acrylic acid

433 243 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(methylsulfonyl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

473 244 (E)-3-(4-((E)-2-(2,4-Dichlorophenyl)-1-(7-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

481 245 (E)-3-(4-((E)-2-(3-Chloropyridin-4-yl)-1-(7-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

448 246 (E)-3-(4-((E)-2-Phenyl-1-(5H-pyrrolo[2,3-b]pyrazin-2-yl)but-1-en-1-yl)phenyl)acrylic acid

396 247 (E)-3-(4-((E)-2-(Benzofuran-5-yl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

435 248 (E)-3-(4-((E)-2-(2-Chloro-3-methoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

459 249 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyrrolidin-1-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

464 250 (E)-3-(4-((E)-2-(2,3- Dihydrobenzo[b][1,4]dioxin-6-yl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

453 251 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(1H-indazol-6-yl)but-1-en-1-yl)phenyl)acrylic acid

435 252 (E)-3-(4-((E)-2-(3-(1H-Pyrazol-1-yl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

461 253 (E)-3-(4-((E)-2-(2,4-Dichlorophenyl)-1-(7-fluoro-1H-indol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

478 254 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(7-fluoro-1H-indol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

Not Observed 255 (E)-3-(4-((E)-2-(Benzo[d]thiazol-5-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

452 256 (E)-3-(4-((E)-2-(2,3-Dihydrobenzofuran-5-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

437 257 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-4-methyl-2-(4-methylthiophen-3-yl)pent-1-en-1- yl)phenyl)acrylic acid

443 258 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(1-methyl-1H-indazol-6-yl)but-1-en-1- yl)phenyl)acrylic acid

449 259 (E)-3-(4-((E)-2-(Benzo[d]thiazol-6-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

452 260 (E)-3-(4-((E)-2-(Imidazo[1,2-a]pyridin-6-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

435 261 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-phenoxyphenyl)but-1-en-1-yl)phenyl)acrylic acid

487 262 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-methyl-3-oxo-3,4-dihydro-2H- benzo[b][1,4]oxazin-6-yl)but-1-en-1-yl)phenyl)acrylic acid

480 263 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-phenoxyphenyl)but-1-en-1-yl)phenyl)acrylic acid

487 264 (E)-3-(4-((E)-2-(4-(1H-Pyrazol-1-yl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

461 265 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-morpholinophenyl)but-1-en-1- yl)phenyl)acrylic acid

480 266 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(quinoxalin-6-yl)but-1-en-1- yl)phenyl)acrylic acid

447 267 (E)-3-(4-((E)-2-(2-Chlorothiophen-3-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

435 268 (E)-3-(4-((E)-2-(Benzo[b]thiophen-2-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

451 269 (E)-3-(4-((E)-2-(3-(4- Fluorophenoxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

505 270 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(5-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)but-1-en-1-yl)phenyl)acrylic acid

470 271 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(5-phenylthiophen-3-yl)but-1-en-1- yl)phenyl)acrylic acid

477 272 (E)-3-(4-((E)-2-(Benzo[b]thiophen-5-yl)-1-(Benzo[b]thiophen-5-yl)-1-

451 273 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methylbenzo[d]thiazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

466 274 (E)-3-(4-((E)-2-(2,5-Dimethylthiophen-3-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

429 275 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-1- yl)phenyl)acrylic acid

464 276 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(4-methylthiophen-3-yl)but-1-en-1- yl)phenyl)acrylic acid

432 277 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-3,3,4,4,4-pentadeutero-1-(1H-indazol-5- yl)but-1-en-1-yl)phenyl)acrylicacid

452 278 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(2-chloro-4-fluorophenyl)-2- cyclobutylvinyl)phenyl)acrylic acid

490 279 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-cyclobutyl-2-(4-methylthiophen-3- yl)vinyl)phenyl)acrylic acid

458 280 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(2-chlorophenyl)but-1-en-1-yl)phenyl)acrylic acid

446 281 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(4-fluorophenyl)but-1-en-1-yl)phenyl)acrylic acid

430 282 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(4-fluoro-2-methylphenyl)but-1-en-1- yl)phenyl)acrylic acid

444 283 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(o-tolyl)but-1-en-1-yl)phenyl)acrylic acid

426 284 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(3-chloropyridin-4-yl)but-1-en-1- yl)phenyl)acrylic acid

447 285 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(3-chloropyridin-4-yl)but-1-en-1- yl)phenyl)acrylic acid

473 286 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-cyclobutyl-2-phenylvinyl)phenyl)acrylic acid

438 287 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-oxoisoindolin-5-yl)but-1-en-1- yl)phenyl)acrylic acid

462 288 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(4-((trifluoromethyl)sulfonyl)phenoxy)phenyl) but-1-en-1-yl)phenyl)acrylicacid

527 289 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(quinolin-7-yl)but-1-en-1- yl)phenyl)acrylic acid

458 290 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyrimidin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

489 291 (E)-3-(4-((E)-2-(2-Chloro-4-phenoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

521 292 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyridin-3-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

488 293 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyrazin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

489 294 (E)-3-(4-((E)-2-(4-Fluoro-3-phenoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

505 295 (E)-3-(4-((E)-2-(3-Fluoro-4-phenoxyphenyl)-1-(1H-indaozl-5-yl)but-1- en-1-yl)phenyl)acrylic acid

505 296 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(4-(pyridin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

506 297 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methyl-5-phenoxyphenyl)but-1-en-1- yl)phenyl)acrylic acid

501 298 (E)-3-(4-((E)-2-(2-Fluoro-3-phenoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

505 299 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(3-phenoxyphenyl)but-1-en-1-yl)phenyl)acrylic acid

504 300 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(3-(pyridin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

505 301 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(3-(pyrazin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

506 302 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-((trifluoromethyl)sulfinyl)phenyl)but-1-en- 1-yl)phenyl)acrylic acid

511 303 (E)-3-(4-((E)-2-(3-Cyanopyridin-4-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

421 304 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(trifluoromethyl)pyridin-4-yl)but-1-en-1- yl)phenyl)acrylic acid

464 305 (E)-3-(4-((E)-2-(3-Chloropyridin-4-yl)-2-cylcobutyl-1-(1H-indazol-5- yl)vinyl)phenyl)acrylic acid

456 306 (E)-3-(4-((E)-2-(3-Chloropyridin-4-yl)-1-(3-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

448 307 (E)-3-(4-((E)-2-(3-Chloropyridin-4-yl)-2-cyclobutyl-1-(3-fluoro-1H-indazol-5- yl)vinyl)phenyl)acrylic acid

474 308 (E)-3-(4-((E)-2-(4-Chloro-2-cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

454 309 (E)-3-(4-((E)-2-(2-Cyano-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

438 310 (E)-3-(4-((E)-2-(2-Cyano-4-(trifluoromethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

488 311 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-2-cyclobutyl-1-(imidazo[1,2-a]pyridin-6- yl)vinyl)phenyl)acrylic acid

473 312 (E)-3-(4-((E)-1-(3-Fluoro-1H-indazol-5-yl)-2-(4-methylthiophen-3-yl)but-1-en-1- yl)phenyl)acrylic acid

433 313 (E)-3-(4-((E)-2-Cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)-2-(4-methylthiophen-3- yl)vinyl)phenyl)acrylic acid

459 314 (E)-3-(4-((E)-2-(Benzo[d]isothiazol-5-yl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

452 315 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyridin-4-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

488 316 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyridin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

488 317 (E)-3-(4-((E)-2-(4-Benzylphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

485 318 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-methyl-3-phenoxyphenyl)but-1-en-1- yl)phenyl)acrylic acid

501 319 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-methyl-4-phenoxyphenyl)but-1-en-1- yl)phenyl)acrylic acid

501 320 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-methyl-4-phenoxyphenyl)but-1-en-1- yl)phenyl)acrylic acid

501 321 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(3-(methylsulfonyl)phenoxy)phenyl)but-1-en- 1-yl)phenyl)acrylic acid

565 322 (E)-3-(4-((E)-2-(2-Chloro-4-cyanophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

454 323 (E)-3-(4-((E)-2-(3-Cyano-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

434 324 (E)-3-(4-((E)-2-(4-Cyano-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

434 325 (E)-3-(4-((E)-2-(5-Cyano-2-methylphenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

434 326 (E)-3-(4-((E)-2-(2-Cyano-4-methoxyphenyl)-1-(1H-indazol-5-yl)but-1- en-1-yl)phenyl)acrylic acid

450 327 (E)-3-(4-((E)-2-(3-(Carboxymethyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

453 328 (E)-3-(4-((E)-2-(4-(Carboxymethyl)phenyl)-1-(1H-indaozl-5-yl)but-1-en-1- yl)phenyl)acrylic acid

453 329 5-((E)-1-(4-((S)-2-((R)-3-Methylpyrrolidin-1-yl)propoxy)phenyl)-2-phenylbut-1-en-1- yl)-1H-indazole

466 330 5-((E)-2-(2-Chlorophenyl)-1-(4-((S)-2-((R)-3-methylpyrrolidin-1- yl)propoxy)phenyl)but-1-en-1-yl)-1H- indazole

500 331 5-((E)-2-(3-Chloropyridin-4-yl)-1-(4-((S)-2-((R)-3-methylpyrrolidin-1- yl)propoxy)phenyl)but-1-en-1-yl)-1H- indazole

501 332 (E)-1-(4-(2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenoxy)cyclopropanecarboxylic acid

477 333 (E)-5-(4-(2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)isoxazol-3-ol

460 334 (E)-3-(4-(2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)isoxazol-5-ol

460 335 (E)-3-(4-((E)-2-(3-Hydroxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

411 336 (E)-3-(4-((E)-2-(2-Hydroxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

411 337 (E)-3-(4-((E)-2-(4-Hydroxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

411 338 (E)-3-(4-(1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)propanoic acid

397 339 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(2-methoxyethoxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

469 340 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(3-methoxypropoxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

483 341 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(2-methoxyethoxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

469 342 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(3-methoxypropoxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

483 343 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(3-((1-methylpiperidin-4-yl)oxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid

526 344 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(3-((tetrahydro-2H-pyran-4- yl)oxy)phenyl)but-1-en-1-yl)phenyl)acrylicacid

513 345 (E)-3-(4-((E)-2-(3-(Cyclohexyloxy)phenyl)-1-(4-fluoro-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

511 346 (E)-3-(4-((E)-2-(3-Butoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

467 347 (E)-3-(4-((E)-2-(3- (Carboxymethoxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

469 348 (E)-3-(4-((E)-2-(4- (Carboxymethoxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

469 349 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pentyloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

481 350 (E)-3-(4-((E)-2-(3-Hexyloxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

495 351 4-(3-((E)-1-(4-((E)-2-Carboxyvinyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-2- yl)phenoxy)butanoic acid

497 352 5-(3-((E)-1-(4-((E)-2-Carboxyvinyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-2- yl)phenoxy)pentanoic acid

511 353 4-(4-((E)-1-(4-((E)-2-Carboxyvinyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-2- yl)phenoxy)butanoic acid

497 354 5-(4-((E)-1-(4-((E)-2-Carboxyvinyl)phenyl)-1-(1H-indazol-5-yl)but-1-en-2- yl)phenoxy)pentanoic acid

511 355 (E)-3-(4-((E)-2-(4-Butoxyphenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

467 356 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pentyloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

481 357 (E)-3-(4-((E)-2-(4-(Hexyloxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

495 358 (E)-3-(4-((E)-2-(3-(Benzyloxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

501 359 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyridin-3-ylmethoxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

502 360 (E)-3-(4-((E)-2-(4-(2- Hydroxyethoxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

455 361 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(2-(methylsulfonyl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

473 362 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyridin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

488 363 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyrimidin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

489 364 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyrimidin-4-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

489 365 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyridin-3-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

488 366 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3- (pyridin-4-yloxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid

488 367 (E)-3-(4-((E)-2-(3-(2- Fluorophenoxy)phenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

505 368 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(o-tolyloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

501 369 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyrimidin-5-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

489 370 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(thiazol-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

494 371 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyrazin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

489 372 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-((3-(trifluoromethyl)pyridin-4- yl)oxy)phenyl)but-1-en-1-yl)phenyl)acrylicacid

556 373 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(4-(methylsulfonyl)phenoxy)phenyl)but-1-en- 1-yl)phenyl)acrylic acid

565 374 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(2-(methylsulfonyl)phenoxy)phenyl)but-1-en- 1-yl)phenyl)acrylic acid

565 375 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(3-(pyridin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

506 376 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(3-(pyrimidin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

507 377 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(3-(pyrazin-2-yloxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

507 378 (E)-3-(4-((E)-1-(4-Fluoro-1H-indazol-5-yl)-2-(3-((6-(methylsulfonyl)pyridin-3-yl)oxy)phenyl)but-1-en-1-yl)phenyl)acrylic acid

584 379 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-((5-(methylsulfonyl)pyridin-2- yl)oxy)phenyl)but-1-en-1-yl)phenyl)acrylicacid

566 380 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-((6-(methylsulfonyl)pyridin-3- yl)oxy)phenyl)but-1-en-1-yl)phenyl)acrylicacid

566 381 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-((5-(trifluoromethyl)pyridin-2- yl)oxy)phenyl)but-1-en-1-yl)phenyl)acrylicacid

556 382 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-((6-methylpyridin-3-yl)oxy)phenyl)but-1-en-1- yl)phenyl)acrylic acid

502 383 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyrimidin-5-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

473 384 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

475 385 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyridin-2-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

472 386 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(thiophen-3-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

477 387 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(thiazol-2-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

478 388 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(thiazol-4-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

478 389 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(pyrazin-2-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

473 390 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyridin-2-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

472 391 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(thiazol-2-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

478 392 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(thiazol-4-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

478 393 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyridin-3-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

472 394 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(3-(pyridin-4-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

472 395 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(isothiazol-4-yl)phenyl)but-1-en-1- yl)phenyl)acrylic acid

478 396 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(methyl(phenyl)amino)phenyl)but-1-en-1- yl)phenyl)acrylic acid

500 397 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-(4-(phenylamino)phenyl)but-1-en-1- yl)phenyl)acrylic acid

486 398 (E)-3-(4-((E)-2-(2-Chlorophenyl)-1-(1-methyl-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

443 399 (E)-3-(4-((E)-2-Cyclobutyl-1-(1-methyl- 1H-indazol-5-yl)-2-phenylvinyl)phenyl)acrylic acid

435 400 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-methyl-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

461 401 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-(difluoromethyl)-1H-indazol-5-yl)but- 1-en-1-yl)phenyl)acrylic acid

497 402 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(2-(difluoromethyl)-2H-indazol-5-yl)but- 1-en-1-yl)phenyl)acrylic acid

497 403 (E)-3-(4-((E)-1-(1-Acetyl-1H-indazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-1- yl)phenyl)acrylic acid

489 404 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-isobutyryl-1H-indazol-5-yl)but-1-en- 1-yl)phenyl)acrylic acid

517 405 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-pivaloyl-1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylic acid

531 406 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-(ethoxycarbonyl)-1H-indazol-5- yl)but-1-en-1-yl)phenyl)acrylic acid

519 407 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylic acid

447 [(M − THP + H) + H]+ 408 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-(methylsulfonyl)acrylamide

524 409 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-(propylsulfonyl)acrylamide

552 410 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-(pentylsulfonyl)acrylamide

580 411 (E)-3-(4-((E)-2-Cyclobutyl-1-(1-methyl-1H-indazol-5-yl)-2-phenylvinyl)phenyl)-N- methylacrylamide

448 412 (E)-3-(4-((E)-2-Cyclobutyl-1-(1H-indazol-5-yl)-2-phenylvinyl)phenyl)-N- methylacrylamide

434 413 (E)-3-(4-((E)-2-Cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)-2-phenylvinyl)phenyl)-N- cyclopropylacrylamide

478 414 (E)-3-(4-((E)-2-Cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)-2-phenylvinyl)phenyl)-N-(2- hydroxyethyl)acrylamide

482 415 (E)-3-(4-((E)-2-Cyclobutyl-1-(3-fluoro-1H-indazol-5-yl)-2-phenylvinyl)phenyl)-N- (2,2,2-trifluoroethyl)acrylamide

520 416 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-1-yl)phenyl)-N-(methylsulfonyl)acrylamide

541 417 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1- yl)phenyl)acrylamide

446 418 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-N- hydroxyacrylamide

410 419 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-N- isopropylacrylamide

436 420 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-methylacrylamide

460 421 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-methoxyacrylamide

476 422 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-methoxy-N-methylacrylamide

490 423 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-N- (methylsulfonyl)acrylamide

472 424 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1-methyl-1H-indazol-5-yl)but-1-en-1-yl)phenyl)-N-(methylsulfonyl)acrylamide

538 425 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-(2H-tetrazol-5-yl)acrylamide

514 426 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylamide

411 427 (E)-3-(4-((E)-1-(Benzo[d]thiazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-N- methylacrylamide

425 428 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)- N-(phenylsulfonyl)acrylamide

586 429 (E)-N-(Benzylsulfonyl)-3-(4-((E)-2-(2-chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylamide

600 430 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-N-(thiazol-2- yl)acrylamide

477 431 (E)-3-(4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)phenyl)-N- cyclopentylacrylamide

462 432 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)-N-(pyridin-2-ylsulfonyl)acrylamide

587 433 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)-N-(pyridin-3-ylsulfonyl)acrylamide

587 434 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)-N-(pyridin-4-ylsulfonyl)acrylamide

587 435 3-((E)-4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)styryl)-1,2,4- oxadiazol-5(4H)-one

435 436 3-((E)-4-((E)-1-(Benzo[d]thiazol-5-yl)-2-(2-chloro-4-fluorophenyl)but-1-en-1-yl)styryl)-1,2,4-oxadiazol-5(4H)-one

504 437 3-((E)-4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)styryl)- 1,2,4-oxadiazol-5(4H)-one

487 438 3-((E)-4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)styryl)-1,2,4- thiadiazol-5(4H)-one

451 439 3-((E)-4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)styryl)- 1,2,4-thiadiazol-5(4H)-one

503 440 3-((E)-4-((E)-1-(1H-Indazol-5-yl)-2-phenylbut-1-en-1-yl)styryl)-1,2,4- oxadiazole-5(4H)-thione

451 441 3-((E)-4-((E)-2-(2-Chloro-4-fluroophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)styryl)- 1,2,4-oxadiazole-5(4H)-thione

503 442 5-((E)-1-(4-((E)-2-(2H-Tetrazol-5-yl)vinyl)phenyl)-2-phenylbut-1-en-1-yl)- 1H-indazole

419 443 5-((E)-1-(4-((E)-2-(2H-Tetrazol-5-yl)vinyl)phenyl)-2-(2-chlorophenyl)but-1- en-1-yl)-1H-indazole

453 444 5-((E)-1-(4-((E)-2-(2H-Tetrazol-5-yl)vinyl)phenyl)-2-phenylbut-1-en-1- yl)benzo[d]thiazole

436 445 5-((E)-1-(4-((E)-2-(2H-Tetrazol-5-yl)vinyl)phenyl)-2-(2-chloro-4- fluorophenyl)but-1-en-1-yl)benzo[d]thiazole

488 446 5-((E)-1-(4-((E)-2-(2H-Tetrazol-5-yl)vinyl)phenyl)-2-(3-chloropyridin-4- yl)but-1-en-1-yl)benzo[d]thiazole

471 447 5-((E)-1-(4-((E)-2-(2H-Tetrazol-5-yl)vinyl)phenyl)-2-(4-methylthiophen-3-yl)but-1-en-1-yl)benzo[d]thiazole

456 448 5-((E)-1-(4-((E)-2-(2HTetrazol-5-yl)vinyl)phenyl)-2-cyclobutyl-2-(4- methylthiophen-3-yl)vinyl)benzo[d]thiazole

482 449 (E)-6-(1-(1H-Indazol-5-yl)-2-phenylbut-1- en-1-yl)-2-naphthoicacid

419 450 (E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(7-hydroxybenzofuran-3-yl)but-1-en-1- yl)phenyl)acrylic acid

463 451 (E)-3-(4-((E)-1-(7-Hydroxybenzofuran-3-yl)-2-phenylbut-1-en-1-yl)phenyl)acrylic acid

411 452 (E)-2-(4-((E)-1-(1H-Indazol-5-yl)-2- phenylbut-1-en-1-yl)phenyl)ethenesulfonamide

430 453 4-((E)-1-(4-((E)-2-Carboxyvinyl)phenyl)-1-(7-fluoro-1H-indazol-5-yl)but-1-en-2- yl)-3-chloropyridine-1-oxide

464

In yet other embodiments, the alternative therapy is a tamoxifenanalogue.

In another embodiment, the alternate therapy is a reduction in the doseof SERM therapy, and/or the addition of an anti-cancer therapy that isnot a SERM. For example, an aromatase inhibitor can be administered inaddition to the lower dose of the SERM therapy. Typically, a subject whois a candidate to receive treatment with an aromatase inhibitor is apost-menopausal female. A premenopausal female is typically not acandidate to receive treatment with an aromatase inhibitor.

In another embodiment, alternative therapies can include administrationof an inhibitor of other steroid receptors, such as inhibitors ofretinoic acid receptors. Retinoic acid receptors (including retinoid Xreceptors) are known to interact with estrogen receptors. Alternativetherapeutic therapies include inhibition of retinoic acid receptoractivity. Therapeutic strategies based on RXR and RAR modulators forcancer treatment are reviewed in Altucci, L. et al. (2007) NatureReviews Drug Discovery 6:793-810.

In one embodiment, a patient, such as a breast cancer patient, receivingtherapy with a SERM (e.g., tamoxifen), can be monitored, e.g., monthly,every two months, or every 3, 4, 5, 6, 7, 8, 9 or 10 months, or yearlyfor the presence of an ESR1 mutation (e.g., an ESR mutation describedherein). In one embodiment, the subject is positive for an estrogenreceptor (e.g., an ER positive subject as detected, e.g., byimmunohistochemistry). If during the course of treatment with a SERM,the patient develops a mutation in the ESR1 domain described herein,e.g., in the vicinity of T311, 5341, A350, G344, D351, R394, Q414, 5433,R503, Y537, and/or D538, the patient can stop treatment with the SERM.In some embodiments, the patient will not stop treatment with the SERMor will be administered a lower dose of the SERM, and optionally, thepatient will receive enhanced monitoring (e.g., more frequentmonitoring) for signs of increased cancer symptoms, e.g., increasedtumor size, metastasis, or spread of the tumor to other organs ortissues.

In one embodiment, the subject, e.g., patient, who develops a mutationin the ESR1 domain, e.g., in the vicinity of T311, 5341, G344, A350,D351, R394, Q414, S433, R503, Y537, and/or D538, is a post-menopausalfemale who will stop treatment with the SERM, and who and will begintreatment with an aromatase inhibitor, an anti-estrogen, a SERD, anestrogen receptor modulator as described herein, fulvestrant (Faslodex®)and/or an mTOR pathway inhibitor.

In yet other embodiments, the subject has one or more mutations inaddition to the mutation of the ESR1 domain. Examples of such mutationscan be found in Table 3, and include a HER2 mutation (e.g., HER2amplification), a p53 mutation, BRCA, NF1, EGFR/myc gains, PIK3CA, CCND1and CDH1. Thus, the subjects can receive a therapy that target the ESR1mutation, alone or in combination with a therapy that targets anothermutation identified as part of the cancer, e.g., a drug that targetsmutant HER2 mutation (e.g., HER2 amplification), a p53 mutation, BRCA,NF1, EGFR/myc gains, PIK3CA, CCND1 and/or CDH1.

The invention provides methods of identifying, assessing or detecting amutant ESR1; methods of identifying, assessing, evaluating, and/ortreating a subject having a cancer, e.g., a cancer having a mutant ESR1;isolated mutant ESR1 nucleic acid molecules, nucleic acid constructs,and host cells containing the nucleic acid molecules; purified mutantESR1 polypeptides and binding agents; detection reagents (e.g., probes,primers, antibodies, kits, e.g., capable of specific detection of amutant ESR1 nucleic acid or protein); screening assays for identifyingmolecules that interact with, e.g., inhibit, mutant ESR1, e.g., novelestrogen receptor inhibitors; as well as assays and kits for evaluating,identifying, assessing and/or treating a subject having a cancer, e.g.,a cancer having a mutant ESR1, e.g., an ESR1 carrying a mutation in theligand binding domain. The compositions and methods identified hereincan be used, for example, to identify new estrogen receptor inhibitors;to evaluate, identify or select a subject, e.g., a patient, having acancer; and to treat or prevent a cancer.

Mutant ESR1 Nucleic Acid Molecules

In one aspect, the invention features a nucleic acid molecule (e.g., anisolated or purified) nucleic acid molecule that includes a fragment ofan ESR1 gene, including the ligand binding domain (also called thehormone binding domain or the AF-2 domain). In embodiments, the fragmentalso includes a DNA binding domain and a hinge domain. In someembodiments, the fragment also includes an AF-1 domain (see FIG. 1). Inone embodiment, the nucleic acid molecule includes an exon of ESR1(e.g., one or more exons encoding a ligand binding domain, a DNA bindingdomain, a hinge domain and an AF-1 domain).

In an embodiment, the mutant ESR1 nucleic acid molecule comprisessufficient ESR1 sequence such that the encoded ESR1 polypeptide hastranscriptional activation activity or DNA binding activity, e.g., haselevated activity, as compared with a reference ESR1, e.g., a wildtypeESR1, e.g., in a cell of a cancer referred to herein. In an embodimentthe encoded mutant ESR1 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 exons from ESR1. The exons are numbered according toRefSeq NM_001122742 (Oct. 2, 2011). In one embodiment, the encodedmutant ESR1 polypeptide, or functional fragment thereof, includes aligand binding domain, a hinge domain, a DNA binding domain, and an AF-1domain.

In one embodiment, the nucleic acid molecule includes a nucleotidesequence that has an in-frame deletion in exon 8 of ESR1. In otherembodiments, the nucleic acid molecule includes a nucleotide sequence inthe region of 152,265,390 to 152,265,600 of chromosome 6. In anotherembodiment, the nucleic acid molecule includes a nucleotide sequencethat includes a breakpoint, e.g., a breakpoint identified in FIGS. 1 and4B. For example, the nucleic acid molecule includes a nucleotidesequence that includes the fusion junction created by the deletion of 6nucleotides in exon 8 of ESR1, e.g., a nucleotide sequence that includesa portion of SEQ ID NO:3 (e.g., a nucleotide sequence within exons 1-12of an ESR1 gene) (e.g., a portion of SEQ ID NO:3 comprising nucleotides1021-1081, 1033-1071, or 1036-1060 of SEQ ID NO:3 (see FIG. 4B)).

In one embodiment, the nucleic acid molecule includes the nucleotidesequence of nucleotides 1-1782 of SEQ ID NO:3 (corresponding to exons 5b(in part) to 12 (in part) of the ESR1 gene), or a fragment thereof, or asequence substantially identical thereto. In another embodiment, thenucleic acid molecule includes the nucleotide sequence of nucleotides553-1782 of SEQ ID NO:3 (e.g., corresponding to exons 6 (in part) to 12(in part) of the ESR1 gene), or a fragment thereof, or a sequencesubstantially identical thereto. In yet other embodiments, the nucleicacid molecule includes the nucleotide sequence shown in FIGS. 4A to 4B(e.g., SEQ ID NO:3) or a fragment thereof, or a sequence substantiallyidentical thereto. In one embodiment, the nucleic acid molecule iscomplementary to at least a portion of a nucleotide sequence disclosedherein, e.g., is capable of hybridizing under a stringency conditiondescribed herein to SEQ ID NO:3 or a fragment thereof. In yet otherembodiments, the nucleic acid molecule hybridizes to a nucleotidesequence that is complementary to at least a portion of a nucleotidesequence disclosed herein, e.g., is capable of hybridizing under astringency condition described herein to a nucleotide sequencecomplementary to SEQ ID NO:3 or a fragment thereof. The nucleotidesequence of a cDNA encoding an exemplary mutant ESR1 is shown in SEQ IDNO:3, and the amino acid sequence is shown in SEQ ID NO:4.

In one embodiment the nucleic acid molecule includes the nucleotidesequence of nucleotides 1 to 1788 of SEQ ID NO:1, and further comprisingone or more of the point mutations in Table 3. In other embodiments, themutation is a nucleotide mutation chosen from one or more of: a C to Treplacement at nucleotide 932; a C to T replacement at nucleotide 1022;a C to A replacement at nucleotide 1049; a G to A replacement atnucleotide 1181A; a C to T replacement at nucleotide 1240; a T to Creplacement at nucleotide 1297; a T to A replacement at nucleotide 1609;an A to G replacement at nucleotide 1610; or an A to G replacement atnucleotide 1613, of SEQ ID NO:1. In one embodiment, the mutation is an Ato G replacement at nucleotide 1613, of SEQ ID NO:1.

In other embodiments, the nucleic acid molecule includes a nucleotidesequence encoding a mutant ESR1 polypeptide that includes a fragment ofa mutant ESR1 gene. In one embodiment, the nucleotide sequence encodes amutant ESR1 gene that includes a ligand binding domain or a functionalfragment thereof. In another embodiment, the nucleotide sequence encodesa fragment of the mutant ESR1 polypeptide including the amino acidsequence of amino acids 1-593 of SEQ ID NO:4 or a fragment thereof, or asequence substantially identical thereto. For example, the nucleic acidmolecule can include a nucleotide sequence encoding a ligand bindingdomain of an ESR1 polypeptide that includes amino acids 312-544 of SEQID NO:4 or a fragment thereof.

In a related aspect, the invention features nucleic acid constructs thatinclude the mutant ESR1 nucleic acid molecules described herein. Incertain embodiments, the nucleic acid molecules are operatively linkedto a native or a heterologous regulatory sequence. Also included arevectors and host cells that include the ESR1 nucleic acid moleculesdescribed herein, e.g., vectors and host cells suitable for producingthe nucleic acid molecules and polypeptides described herein.

In a related aspect, methods of producing the nucleic acid molecules andpolypeptides described herein are also described.

In another aspect, the invention features nucleic acid molecules thatreduce or inhibit the expression of a nucleic acid molecule that encodesa mutant ESR1 described herein. Examples of such nucleic acid moleculesinclude, for example, antisense molecules, ribozymes, RNAi, triple helixmolecules that hybridize to a nucleic acid encoding a mutant ESR1, or atranscription regulatory region of mutant ESR1, and blocks or reducesmRNA expression of mutant ESR1.

In one aspect, the invention features a nucleic acid molecule (e.g., anisolated or purified) that includes a mutation identified in Table 1 orTable 2.

Nucleic Acid Detection and Capturing Reagents

The invention also features a nucleic acid molecule, e.g., nucleic acidfragment, suitable as probe, primer, bait or library member thatincludes, flanks, hybridizes to, and which are useful for identifying,or are otherwise based on, the ESR1 mutants described herein. In certainembodiments, the probe, primer or bait molecule is an oligonucleotidethat allows capture, detection or isolation of a mutant ESR1 nucleicacid molecule described herein. The oligonucleotide can include anucleotide sequence substantially complementary to a fragment of an ESR1nucleic acid molecule described herein. The sequence identity betweenthe nucleic acid fragment, e.g., the oligonucleotide, and the targetmutant ESR1 sequence need not be exact, so long as the sequences aresufficiently complementary to allow the capture, detection or isolationof the target sequence. In one embodiment, the nucleic acid fragment isa probe or primer that includes an oligonucleotide between about 5 and25, e.g., between 10 and 20, or 10 and 15 nucleotides in length. Inother embodiments, the nucleic acid fragment is a bait that includes anoligonucleotide between about 100 to 300 nucleotides, 130 and 230nucleotides, or 150 and 200 nucleotides, in length.

In one embodiment, the nucleic acid fragment can be used to identify orcapture, e.g., by hybridization, a mutant ESR1. For example, the nucleicacid fragment can be a probe, a primer, or a bait, for use inidentifying or capturing, e.g., by hybridization, a mutant ESR1described herein. In one embodiment, the nucleic acid fragment can beuseful for identifying or capturing an ESR1 breakpoint, e.g., asidentified in FIG. 1 and in FIG. 4B. In one embodiment, the nucleic acidfragment hybridizes to a nucleotide sequence within a chromosomaldeletion of three nucleotides or more that creates an in-frame fusion ofnucleotides within exon 8 of ESR1 (e.g., a sequence within chromosome 6,at exon 8 of ESR1). In one embodiment, the nucleic acid fragmenthybridizes to a nucleotide sequence in the region of152,265,590-152,265,600 of chromosome 6. In one embodiment, the nucleicacid fragment hybridizes to a nucleotide sequence that includes abreakpoint, e.g., a breakpoint as identified in FIGS. 1 and 4B. Forexample, the nucleic acid fragment can hybridize to a nucleotidesequence that includes the fusion junction created by the deletion of 6nucleotides in exon 8 of ESR1, e.g., a nucleotide sequence that includesa portion of SEQ ID NO:3 (e.g., a nucleotide sequence within exons 1-12of an ESR1 gene) (e.g., a portion of SEQ ID NO:3 comprising nucleotides1021-1081, 1033-1071, or 1036-1060 of SEQ ID NO:3 (see FIG. 4B)).

The probes or primers described herein can be used, for example, forFISH detection or PCR amplification. In one exemplary embodiment wheredetection is based on PCR, amplification of an ESR1 mutation, such as adeletion junction in the ligand binding domain, can be performed using aprimer or a primer pair, e.g., for amplifying a sequence flanking theESR1 deletion junction described herein. In one embodiment, a pair ofisolated oligonucleotide primers can amplify a region containing oradjacent to an ESR1 mutation. For example, forward primers can bedesigned to hybridize to a nucleotide sequence within ESR1 genomic ormRNA sequence (e.g., a nucleotide sequence within exons 1-8 of an ESR1gene, or nucleotides 1-1053 of SEQ ID NO:3), and the reverse primers canbe designed to hybridize to a nucleotide sequence within ESR1 (e.g., anucleotide sequence within exons 8-12 of ESR1, or nucleotides 1036-1782of SEQ ID NO:3).

In another embodiment, the nucleic acid fragments can be used toidentify, e.g., by hybridization, a mutant ESR1. In one embodiment, thenucleic acid fragment hybridizes to a nucleotide sequence that includesa fusion junction created by a deletion in exon 8 of an ESR1 transcript,e.g., a nucleotide sequence within SEQ ID NO:3 (e.g., a sequencecomprising nucleotides 1021-1081, 1033-1071, or 1036-1060 of SEQ ID NO:3(see FIG. 4B)). In other embodiments, the nucleic acid fragment detects(e.g., hybridizes to) a nucleotide mutation chosen from one or more of:a C to T replacement at nucleotide 932; a C to T replacement atnucleotide 1022; a C to A replacement at nucleotide 1049; a G to Areplacement at nucleotide 1181A; a C to T replacement at nucleotide1240; a T to C replacement at nucleotide 1297; a T to A replacement atnucleotide 1609; an A to G replacement at nucleotide 1610; or an A to Greplacement at nucleotide 1613, of SEQ ID NO:1. In one embodiment, themutation is an A to G replacement at nucleotide 1613, of SEQ ID NO:1.

In other embodiments, the nucleic acid fragment includes a bait thatcomprises a nucleotide sequence that hybridizes to a mutant ESR1 nucleicacid molecule described herein, and thereby allows the capture orisolation of said nucleic acid molecule. In one embodiment, a bait issuitable for solution phase hybridization. In other embodiments, a baitincludes a binding entity, e.g., an affinity tag, that allows captureand separation, e.g., by binding to a binding entity, of a hybrid formedby a bait and a nucleic acid hybridized to the bait.

In other embodiments, the nucleic acid fragment includes a librarymember comprising a mutant ESR1 nucleic acid molecule described herein.In one embodiment, the library member includes a non-frameshift deletionin the ligand binding domain of ESR1.

The nucleic acid fragment can be detectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label, or caninclude an affinity tag; a tag, or identifier (e.g., an adaptor, barcodeor other sequence identifier).

Mutant ESR1 Polypeptides

In another aspect, the invention features a mutant ESR1 polypeptide(e.g., a purified mutant ESR1 polypeptide), a biologically active orantigenic fragment thereof, as well as reagents (e.g., antibodymolecules that bind to a mutant ESR1 polypeptide), methods formodulating a mutant ESR1 polypeptide activity and detection of a mutantESR1 polypeptide.

In one embodiment, the mutant ESR1 polypeptide has at least onebiological activity, e.g., a ligand binding activity, transcriptionalactivation activity and/or a DNA binding activity. These activities caninclude interaction with the ESR1 mutant polypeptide with coactivatorssuch as Tif2, Src-1, and Grip1. In one embodiment, at least onebiological activity of a mutant ESR1 polypeptide is reduced or inhibitedby an anti-cancer drug, e.g., estrogen inhibitor, such as a SERM or anaromatase inhibitor, a SERD or an estrogen receptor modulator asdescribed herein.

In other embodiments, the mutant ESR1 polypeptide includes a fragment ofa mutant ESR1 polypeptide containing a mutation in the ligand bindingdomain. In one embodiment, the mutant ESR1 polypeptide includes aminoacids 340-360 of SEQ ID NO:4 or a fragment thereof (e.g., amino acids1-593 of SEQ ID NO:4 or a fragment thereof). In yet other embodiments,the mutant ESR1 polypeptide includes an amino acid sequencesubstantially identical to a non-frameshift deletion of amino acids349-351 of SEQ ID NO:2 or a fragment thereof. In other embodiments, themutation is a missense mutation. In one embodiment, the missensemutation is chosen from one or more of: a threonine to methioninesubstitution at position 311 (a T311M); a serine to leucine substitutionat position 341 (a S341L); an alanine to glutamate substitution atposition 350 (a A350E); an arginine to histidine substitution atposition 394 (a R394H); a glutamine substitution at position 414, e.g.,an insertion to a stop codon (a Q414*); a serine to proline substitutionat position 433 (a S433P); an arginine to tryptophan substitution atposition 503 (a R503W); a tyrosine to asparagine substitution atposition 537 (a Y537N); a tyrosine to cysteine substitution at position537 (a Y537C); or an aspartate to glycine substitution at position 538(a D538G), of SEQ ID NO:2. In other embodiment, the mutation is aninsertion mutation, e.g., an amino acid insertion between amino acidsG344 and L345 of SEQ ID NO:2, e.g., an insertion of a C amino acid.

In other embodiments, the mutant ESR1 polypeptide includes a ligandbinding domain or a fragment thereof, a hinge region or a fragmentthereof, a DNA binding domain or fragment thereof, and an AF-1 domain orfragment thereof. In another embodiment, the mutant ESR1 polypeptideincludes the amino acid sequence of amino acids 1-593 of SEQ ID NO:4 ora fragment thereof, or a sequence substantially identical thereto. Forexample, the mutant ESR1 polypeptide can include a ligand binding domainof ESR1 that includes amino acids 312-544 of SEQ ID NO:4 or a fragmentthereof. In other embodiments, the mutant ESR1 polypeptide includes theamino acid sequence of amino acids 185-593 of SEQ ID NO:4 or a fragmentthereof, or a sequence substantially identical thereto.

In yet other embodiments, the mutant ESR1 polypeptide is encoded by anucleic acid molecule described herein. In one embodiment, the mutantESR1 polypeptide is encoded by nucleic acid having a non-frameshiftdeletion in exon 8 of ESR1, or a missense mutation as described herein.In other embodiments, the mutant ESR1 polypeptide is encoded by anucleotide sequence in the region of 152,265,560-152,265,630 ofchromosome 6. In another embodiment, the mutant ESR1 polypeptideincludes an amino acid sequence encoded by a nucleotide sequencecomprising a fusion junction created by a deletion in the ligand bindingdomain of ESR1 transcript, e.g., a nucleotide sequence that includes aportion of SEQ ID NO:3 (e.g., a nucleotide sequence within exons 1-12 ofan ESR1 gene) (e.g., a portion of SEQ ID NO:3 comprising nucleotides1021-1081, 1033-1071, or 1036-1060 of SEQ ID NO:3 (see FIG. 4B)). Inother embodiment, the mutant ESR1 polypeptide is encoded by a nucleicacid that includes a nucleotide mutation chosen from one or more of: a Cto T replacement at nucleotide 932; a C to T replacement at nucleotide1022; a C to A replacement at nucleotide 1049; a G to A replacement atnucleotide 1181A; a C to T replacement at nucleotide 1240; a T to Creplacement at nucleotide 1297; a T to A replacement at nucleotide 1609;an A to G replacement at nucleotide 1610; or an A to G replacement atnucleotide 1613, of SEQ ID NO:1. In one embodiment, the mutation is an Ato G replacement at nucleotide 1613, of SEQ ID NO:1. In yet otherembodiments, the mutant ESR1 polypeptide is encoded by a nucleic acidthat includes an insertion of nucleotides GCT between nucleotides 1032and 1033 of SEQ ID NO:1.

In an embodiment, the mutant ESR1 polypeptide comprises sufficient ESR1sequence such that it has ligand binding activity, e.g., has elevated orconstitutive activity. In one embodiment, the mutant ESR1 polypeptidebinds estrogen with a higher affinity, as compared with wildtype ESR1e.g., in a cell of a cancer referred to herein. In an embodiment, themutant ESR1 polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 or 12 exons, e.g., exons 5b, 6, 7, 8, 9, 10, 11 and 12, of amutant ESR1 transcript. In one embodiment, the mutant ESR1 polypeptide,or a functional fragment thereof, includes a ligand binding domain or afunctional fragment thereof, a hinge domain or a functional fragmentthereof, a DNA binding domain or a functional fragment thereof and anAF-1 domain or a functional fragment thereof. In a related aspect, theinvention features a mutant ESR1 polypeptide or fragments operativelylinked to heterologous polypeptides to form fusion proteins.

In another embodiment, the mutant ESR1 polypeptide or fragment thereofis a peptide, e.g., an immunogenic peptide or protein that contains afusion junction created by a deletion mutant, or a missense mutationdescribed herein. Such immunogenic peptides or proteins can be used toraise antibodies specific to the mutant protein. In other embodiments,such immunogenic peptides or proteins can be used for vaccinepreparation. The vaccine preparation can include other components, e.g.,an adjuvant.

In another aspect, the invention features antibody molecules that bindto a mutant ESR1 polypeptide or fragment described herein. Inembodiments the antibody can distinguish wild-type ESR1 from mutantESR1.

In one aspect, the invention features a polypeptide comprising amutation identified in Table 1 or Table 2 or Table 3.

Methods for Detecting Mutant ESR1

In another aspect, the invention features a method of determining thepresence of a mutant ESR1, e.g., a mutant ESR1 as described herein. Inone embodiment, the mutant ESR1 is detected in a nucleic acid moleculeor a polypeptide. The method includes detecting whether a mutant ESR1nucleic acid molecule or polypeptide is present in a cell (e.g., acirculating cell), a tissue (e.g., a tumor), or a sample, e.g., a tumorsample, from a subject. In one embodiment, the sample is a nucleic acidsample. In one embodiment, the nucleic acid sample comprises DNA, e.g.,genomic DNA or cDNA, or RNA, e.g., mRNA. In other embodiments, thesample is a protein sample.

In one embodiment, the sample is, or has been, classified asnon-malignant using other diagnostic techniques, e.g.,immunohistochemistry.

In one embodiment, the sample is, or has been, classified as malignantusing other diagnostic techniques, e.g., immunohistochemistry. In oneembodiment, the sample is ER-positive.

In one embodiment, the sample is acquired from a subject (e.g., asubject, e.g., a patient, having or at risk of having a cancer, e.g., apatient), or alternatively, the method further includes acquiring asample from the subject. The sample can be chosen from one or more of:tissue, e.g., cancerous tissue (e.g., a tissue biopsy), whole blood,serum, plasma, buccal scrape, sputum, saliva, cerebrospinal fluid,urine, stool, circulating tumor cells, circulating nucleic acids, orbone marrow. In certain embodiments, the sample is a tissue (e.g., atumor biopsy), a circulating tumor cell or nucleic acid.

In embodiments, the tumor is from a cancer described herein, e.g., ischosen from a breast cancer, a prostate cancer, an ovarian cancer, anendometrial cancer or a colon cancer or a combination thereof.

In one embodiment, the ESR1 is detected in a patient whose cancer hasrecurred, such as after a period of remission. In another embodiment,the mutant ESR1 is detected in a metastasis. For example, a breastcancer patient receiving, or having previously received, an anti-cancertreatment such as a SERM, e.g., tamoxifen, can have a cancer, e.g. abreast cancer, that recurs or that has metastasized (e.g., an ER+ breastcancer patient with late stage, progressive metastatic cancer). Themutant ESR1 can be detected in a circulating tumor cell, or in ametastasis, such as in a metastasis from a tissue other than the tissueof the primary tumor (e.g., in a tissue other than breast tissue, suchas from blood, bone, lung or liver tissue). The patient can continue tobe administered the SERM, or can receive a lower dose of a SERM after amutation in the ligand binding domain of ESR1 is detected.Alternatively, the patient can be administered an anti-cancer agent thatis not a SERM. For example, in one embodiment, the patient is apost-menopausal female who is not administered a SERM, and who isadministered an aromatase inhibitor, a SERD, a fulvestrant, or otherestrogen receptor modulator described herein.

In one embodiment, the subject is at risk of having, or has a cancer(e.g., a patient with a cancer described herein).

In other embodiments, the mutant ESR1 is detected in a nucleic acidmolecule by a method chosen from one or more of: nucleic acidhybridization assay, amplification-based assays (e.g., polymerase chainreaction (PCR)), PCR-RFLP assay, real-time PCR, sequencing, screeninganalysis (including metaphase cytogenetic analysis by standard karyotypemethods, FISH (e.g., break away FISH), spectral karyotyping or MFISH,comparative genomic hybridization), in situ hybridization, SSP, HPLC ormass-spectrometric genotyping.

In one embodiment, the method includes: contacting a nucleic acidsample, e.g., a genomic DNA sample (e.g., a chromosomal sample or afractionated, enriched or otherwise pre-treated sample) or a geneproduct (mRNA, cDNA), obtained from the subject, with a nucleic acidfragment (e.g., a probe or primer as described herein (e.g., anexon-specific probe or primer) under conditions suitable forhybridization, and determining the presence or absence of the mutantESR1 nucleic acid molecule. The method can, optionally, includeenriching a sample for the gene or gene product.

In a related aspect, a method for determining the presence of a mutantESR1 nucleic acid molecule is provided. The method includes: acquiring asequence for a position in a nucleic acid molecule, e.g., by sequencingat least one nucleotide of the nucleic acid molecule (e.g., sequencingat least one nucleotide in the nucleic acid molecule that comprises themutant ESR1), thereby determining that the mutant ESR1 is present in thenucleic acid molecule. Optionally, the sequence acquired is compared toa reference sequence, or a wild type reference sequence. In oneembodiment, the nucleic acid molecule is from a cell (e.g., acirculating cell), a tissue (e.g., a tumor), or any sample from asubject (e.g., blood or plasma sample). In other embodiments, thenucleic acid molecule from a tumor sample (e.g., a tumor or cancersample) is sequenced. In one embodiment, the sequence is determined by anext generation sequencing method. The method further can furtherinclude acquiring, e.g., directly or indirectly acquiring, a sample,e.g., a tumor or cancer sample, from a subject (e.g., a patient). Incertain embodiments, the cancer is chosen from a breast cancer, prostatecancer, ovarian cancer, endometrial cancer, or colon cancer.

In another aspect, the invention features a method of analyzing a tumoror a circulating tumor cell. The method includes acquiring a nucleicacid sample from the tumor or the circulating cell; and sequencing,e.g., by a next generation sequencing method, a nucleic acid molecule,e.g., a nucleic acid molecule that includes a mutant ESR1 describedherein. In one embodiment the invention features a method of analyzing ametastasis, e.g., in a tissue separate from the site of the primarytumor.

In yet other embodiments, a mutant ESR1 polypeptide is detected. Themethod includes: contacting a protein sample with a reagent whichspecifically binds to a mutant ESR1 polypeptide; and detecting theformation of a complex of the mutant ESR1 polypeptide and the reagent.In one embodiment, the reagent is labeled with a detectable group tofacilitate detection of the bound and unbound reagent. In oneembodiment, the reagent is an antibody molecule, e.g., is selected fromthe group consisting of an antibody, and antibody derivative, and anantibody fragment.

In yet another embodiment, the level (e.g., expression level) oractivity of the mutant ESR1 is evaluated. For example, the level (e.g.,expression level) or activity of the mutant ESR1 (e.g., mRNA orpolypeptide) is detected and (optionally) compared to a pre-determinedvalue, e.g., a reference value (e.g., a control sample).

In yet another embodiment, the mutant ESR1 is detected prior toinitiating, during, or after, a treatment in a subject, e.g., atreatment with an estrogen inhibitor, e.g., a SERM, an aromataseinhibitor, a SERD, or other estrogen receptor modulator disclosedherein. In one embodiment, the mutant ESR1 is detected at the time ofdiagnosis with a cancer. In other embodiments, the mutant ESR1 isdetected at a pre-determined interval, e.g., a first point in time andat least at a subsequent point in time.

In certain embodiments, responsive to a determination of the presence ofthe mutant ESR1, the method further includes one or more of:

(1) stratifying a patient population (e.g., assigning a subject, e.g., apatient, to a group or class);

(2) identifying or selecting the subject as likely or unlikely torespond to a treatment, e.g., a SERM inhibitor treatment as describedherein;

(3) selecting a treatment option, e.g., administering or notadministering a preselected therapeutic agent, e.g., a SERM or anaromatase inhibitor as described herein; or

(4) prognosticating the time course of the disease in the subject (e.g.,evaluating the likelihood of increased or decreased patient survival).

In certain embodiments, the estrogen inhibitor is an aromatase inhibitoror a SERM. In one embodiment, the aromatase inhibitor is chosen fromaminoglutethimide, testolactone (Teslac®), anastrozole (Arimidex®),letrozole (Femara®), exemestane (Aromasin®), vorozole (Rivizor),formestane (Lentaron®), fadrozole (Afema); 4-hydroxyandrostenedione,1,4,6-androstatrien-3,17-dione (ATD), and 4-Androstene-3,6,17-trione(“6-OXO”). In other embodiments, a SERM is chosen from raloxifene(Evista®), EM652, GW7604, keoxifene, toremifene (Fareston®), tamoxifen(Nolvadex®), lasofoxifene, levormeloxifene, bazedoxifene, or arzoxifene.In another embodiment the anti-estrogen agent is the estrogen antagonistis fulvestrant (ICI 182, 780; Faslodex®). In other embodiment, theanti-estrogen agent is a SERD. In yet other embodiments, theanti-estrogen agent is described in, e.g., WO 2012/037411, WO2012/037410, WO 2011/159769, WO 2011/156518 and US 2012/0071535.

In one embodiment, the mTOR pathway inhibitor is chosen from rapamycin,temsirolimus (TORISEL®), everolimus (RAD001, AFINITOR®), ridaforolimus,AP23573, AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126,OSI-027, GSK1059615, KU-0063794, WYE-354, INK128, temsirolimus(CCI-779), Palomid 529 (P529), PF-04691502, or PKI-587.

In certain embodiments, responsive to the determination of the presenceof the mutant ESR1, the subject is classified as a candidate to receivetreatment with an anti-cancer agent that is not a SERM. For example, thesubject is a post-menopausal female classified as a candidate to receivetreatment with an aromatase inhibitor. In one embodiment, the subject isa pre-menopausal female classified as a candidate to receive treatmentwith an estrogen receptor blocking agent, or to receive an oophorectomy.In another embodiment, the subject is classified as a candidate tocontinue receiving treatment with the SERM, and optionally, to receivemore frequent monitoring for worsening cancer symptoms, e.g., increasedtumor size, metastasis, or appearance of the cancer in additionaltissues.

In one embodiment, responsive to the determination of the presence ofthe mutant ESR1, the subject, e.g., a patient, can further be assignedto a particular class if a mutant ESR1 is identified in a sample of thepatient. For example, a post-menopausal female patient identified ashaving a mutant ESR1 can be classified as a candidate to receivetreatment with an aromatase inhibitor, e.g., an aromatase inhibitor, aSERD, an estrogen receptor modulator, or an mTOR pathway inhibitor, asdescribed herein. In one embodiment, the subject, e.g., a patient, isassigned to a second class if the mutation is not present. For example,a patient who has a breast cancer that does not contain a mutant ESR1,may be determined to be a candidate to receive a SERM, or to continuereceiving a SERM, such as tamoxifen, alone or in combination with otheragents, e.g., an aromatase inhibitor, a SERD, an estrogen receptormodulator, or an mTOR pathway inhibitor.

In another embodiment, responsive to the determination of the presenceof the mutant ESR1, the subject is identified as likely to respond to atreatment that comprises an aromatase inhibitor e.g., an aromataseinhibitor, a SERD, an estrogen receptor modulator, or an mTOR pathwayinhibitor, as described herein. Typically, a subject identified as beinglikely to respond to the treatment is a post-menopausal female.

In yet another embodiment, responsive to the determination of thepresence of the mutant ESR1, the method includes administering anaromatase inhibitor, a SERD, an estrogen receptor modulator, or an mTORpathway inhibitor, as described herein, to the subject.

In one embodiment, a subject who is determined not to carry a mutantESR1 is reevaluated at intervals, such as every month, every two months,every six months or every year, or more or less frequently, to monitorthe patient for the development of a mutation in ESR1, e.g., in theligand binding domain of ESR1. For example, if a patient is determinednot to carry a mutant ESR1, then the patient can be determined to be acandidate for treatment with a SERM. If the patient is subsequentlydetermined to have a mutant ESR1, administration of the SERM to thepatient can be stopped, and the patient can be administered ananti-cancer agent that is not a SERM. For example, the patient may be apost-menopausal female who is administered an aromatase inhibitor or anmTOR pathway inhibitor, or the patient may be a pre-menopausal femalewho is administered an alternative estrogen receptor blocking agent orwho is administered an oophorectomy. In some embodiments, the patientcontinues to receive treatment with the SERM (e.g., tamoxifen), andoptionally, the patient can receive more frequent monitoring for aworsening of cancer symptoms.

In one embodiment, a patient determined to have a mutant ESR1, e.g., amutation in the ligand binding domain of ESR1, is administered a lowerdose of the SERM, and optionally, a second anti-cancer agent, such as anaromatase inhibitor, a SERD, an estrogen receptor modulator, or an mTORpathway inhibitor.

Detection Reagents and Detection of Mutations

In another aspect, the invention features a detection reagent, e.g., apurified or isolated preparation thereof. Detection reagents candistinguish a nucleic acid, or protein sequence, having a mutation,e.g., a missense, or in-frame insertion or deletion, in ESR1, e.g., inthe ligand binding or hinge region of ESR1, from a reference sequence.In an embodiment, the mutation is a mutation described herein, e.g., anyof: a 6 nucleotide deletion of nucleotides 1046-1051 (TGGCAG) accordingto SEQ ID NO:1 or other deletion, e.g., an in-frame deletion, thatincludes one or more of nucleotides 1046-1051; a mutation at a positionidentified as mutated in Table 3, e.g., any of the mutations describedin Table 3; or, an associated mutation. Detection reagents, e.g.,nucleic acid-based detection reagents, can be used to identify mutationsin a target nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA,e.g., in a sample, e.g., a sample of nucleic acid derived from a tumorcell, e.g., an ER⁺ tumor cell. Detection reagents, e.g., antibody-baseddetection reagents, can be used to identify mutations in a targetprotein, e.g., in a sample, e.g., a sample of protein derived from, orproduced by, a tumor cell.

Nucleic Acid-Based Detection Reagents

In an embodiment, the detection reagent comprises a nucleic acidmolecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, comprisingsequence which is complementary with a nucleic acid sequence on a targetnucleic acid (the sequence on the target nucleic acid that is bound bythe detection reagent is referred to herein as the “detection reagentbinding site” and the portion of the detection reagent that correspondsto the detection reagent binding site is referred to as the “targetbinding site”). In an embodiment the detection reagent binding site isdisposed in relationship to the interrogation position such that binding(or in embodiments, lack of binding) of the detection reagent to thedetection reagent binding site allows differentiation of mutant andreference sequences for a mutant described herein. The detection reagentcan be modified, e.g., with a label or other moiety, e.g., a moiety thatallows capture.

In an embodiment, the detection reagent comprises a nucleic acidmolecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, which, e.g., inits target binding site, includes the interrogation position and whichcan distinguish (e.g., by affinity of binding of the detection reagentto a target nucleic acid or the ability for a reaction, e.g., a ligationor extension reaction with the detection reagent) between a mutation,e.g., a mutation described herein, and a reference sequence. Inembodiments, the interrogation position can correspond to a terminal,e.g., to a 3′ or 5′ terminal nucleotide, a nucleotide immediatelyadjacent to a 3′ or 5′ terminal nucleotide, or to another internalnucleotide, of the detection reagent or target binding site.

In embodiments, the difference in the affinity of the detection reagentfor a target nucleic acid comprising the mutant and that for a targetnucleic acid comprising the reference sequence allows determination ofthe presence or absence of the mutation (or reference) sequence.Typically such detection reagents, under assay conditions, will exhibitsubstantially higher levels of binding only to the mutant or only to thereference sequence, e.g., will exhibit substantial levels of bindingonly to the mutant or only to the reference sequence.

In embodiments, binding allows (or inhibits) a subsequent reaction,e.g., a subsequent reaction involving the detection reagent or thetarget nucleic acid. E.g., binding can allow ligation, or the additionof one or more nucleotides to a nucleic acid, e.g., the detectionreagent, e.g., by DNA polymerase, which can be detected and used todistinguish mutant from reference. In embodiments, the interrogationposition is located at the terminus, or sufficiently close to theterminus, of the detection reagent or its target binding site, such thathybridization, or a chemical reaction, e.g., the addition of one or morenucleotides to the detection reagent, e.g., by DNA polymerase, onlyoccurs, or occurs at a substantially higher rate, when there is aperfect match between the detection reagent and the target nucleic acidat the interrogation position or at a nucleotide position within 1, 2,or 3 nucleotides of the interrogation position.

In an embodiment, the detection reagent comprises a nucleic acid, e.g.,a DNA, RNA or mixed DNA/RNA molecule wherein the molecule, or its targetbinding site, is adjacent (or flanks), e.g., directly adjacent, to theinterrogation position, and which can distinguish between a mutation,e.g., a mutation described herein, and a reference sequence, in a targetnucleic acid.

In embodiments the detection reagent binding site is adjacent to theinterrogation position, e.g., the 5′ or 3′terminal nucleotide of thedetection reagent, or its target binding site, is adjacent, e.g.,between 0 (directly adjacent) and 1,000, 500, 400, 200, 100, 50, 10, 5,4, 3, 2, or 1 nucleotides from the interrogation position. Inembodiments, the outcome of a reaction will vary with the identity ofthe nucleotide at the interrogation position allowing one to distinguishbetween mutant and reference sequences. E.g., in the presence of a firstnucleotide at the interrogation position a first reaction will befavored over a second reaction. E.g., in a ligation or primer extensionreaction, the product will differ, e.g., in charge, sequence, size, orsusceptibility to a further reaction (e.g., restriction cleavage)depending on the identity of the nucleotide at the interrogationposition. In embodiments the detection reagent comprises pairedmolecules (e.g., forward and reverse primers), allowing foramplification, e.g., by PCR amplification, of a duplex containing theinterrogation position. In such embodiments, the presence of themutation can be determined by a difference in the property of theamplification product, e.g., size, sequence, charge, or susceptibilityto a reaction, resulting from a sequence comprising the interrogationposition and a corresponding sequence having a reference nucleotide atthe interrogation positions. In embodiments, the presence or absence ofa characteristic amplification product is indicative of the identity ofthe nucleotide at the interrogation site and thus allows detection ofthe mutation.

In embodiments the detection reagent, or its target binding site, isdirectly adjacent to the interrogation position, e.g., the 5′ or3′terminal nucleotide of the detection reagent is directly adjacent tothe interrogation position. In embodiments, the identity of thenucleotide at the interrogation position will determine the nature of areaction, e.g., a reaction involving the detection reagent, e.g., themodification of one end of the detection reagent. E.g., in the presenceof a first nucleotide at the interrogation position a first reactionwill be favored over a second reaction. By way of example, the presenceof a first nucleotide at the interrogation position, e.g., a nucleotideassociated with a mutation, can promote a first reaction, e.g., theaddition of a complementary nucleotide to the detection reagent. By wayof example, the presence of an A at the interrogation position willcause the incorporation of a T, having e.g., a first colorometric label,while the presence of a G and the interrogation position will cause theincorporation for a C, having, e.g., a second colorometric label. In anembodiment the presence of a first nucleotide at the nucleotide willresult in ligation of the detection reagent to a second nucleic acid.E.g., a third nucleic acid can be hybridized to the target nucleic acidsufficiently close to the interrogation site that if the third nucleicacid has an exact match at the interrogation site it will be ligated tothe detection reagent. Detection of the ligation product, or itsabsence, is indicative of the identity of the nucleotide at theinterrogation site and thus allows detection of the mutation.

A variety of readouts can be employed. E.g., binding of the detectionreagent to the mutant or reference sequence can be followed by a moiety,e.g., a label, associated with the detection reagent, e.g., aradioactive or enzymatic label. In embodiments the label comprises aquenching agent and a signaling agent and hybridization results inaltering the distance between those two elements, e.g., increasing thedistance and unquenching the signaling agent. In embodiments thedetection reagent can include a moiety that allows separation from othercomponents of a reaction mixture. In embodiments binding allows cleavageof the bound detection reagent, e.g., by an enzyme, e.g., by thenuclease activity of the DNA polymerase or by a restriction enzyme. Thecleavage can be detected by the appearance or disappearance of a nucleicacid or by the separation of a quenching agent and a signaling agentassociated with the detection reagent. In embodiments binding protects,or renders the target susceptible, to further chemical reaction, e.g.,labeling or degradation, e.g., by restriction enzymes. In embodimentsbinding with the detection reagent allows capture separation or physicalmanipulation of the target nucleic acid to thereby allow foridentification. In embodiments binding can result in a detectablelocalization of the detection reagent or target, e.g., binding couldcapture the target nucleic acid or displace a third nucleic acid.Binding can allow for determination of the presence of mutant orreference sequences with FISH, particularly in the case ofrearrangements. Binding can allow for the extension or other size changein a component, e.g., the detection reagent, allowing distinctionbetween mutant and reference sequences. Binding can allow for theproduction, e.g., by PCR, of an amplicon that distinguishes mutant fromreference sequence.

In an embodiment the detection reagent, or the target binding site, isbetween 5 and 500, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and100, 5 and 50, 5 and 25, 5 and 20, 5 and 15, or 5 and 10 nucleotides inlength. In an embodiment the detection reagent, or the target bindingsite, is between 10 and 500, 10 and 300, 10 and 250, 10 and 200, 10 and150, 10 and 100, 10 and 50, 10 and 25, 10 and 20, or 10 and 15,nucleotides in length. In an embodiment the detection reagent, or thetarget binding site, is between 20 and 500, 20 and 300, 20 and 250, 20and 200, 20 and 150, 20 and 100, 20 and 50, or 20 and 25 nucleotides inlength. In an embodiment the detection reagent, or the target bindingsite, is sufficiently long to distinguish between mutant and referencesequences and is less than 100, 200, 300, 400, or 500 nucleotides inlength.

Preparations of Mutant Nucleic Acid and Uses Thereof

In another aspect the invention features, purified or isolatedpreparations of a an ER⁺ tumor cell nucleic acid, e.g., an ESR1 nucleicacid, e.g., DNA, e.g., genomic DNA or cDNA, or RNA, containing aninterrogation position described herein, useful for determining if amutation disclosed herein is present. The nucleic acid includes theinterrogation position, and typically additional ER1 sequence on one orboth sides of the interrogation position. In addition the nucleic acidcan contain heterologous sequences, e.g., adaptor or priming sequences,typically attached to one or both terminus of the nucleic acid. Thenucleic acid also includes a label or other moiety, e.g., a moiety thatallows separation or localization.

In embodiments, the nucleic acid is between 20 and 1,000, 30 and 900, 40and 800, 50 and 700, 60 and 600, 70 and 500, 80 and 400, 90 and 300, or100 and 200 nucleotides in length (with or without heterologoussequences). In embodiment the nucleic acid is between 40 and 1,000, 50and 900, 60 and 800, 70 and 700, 80 and 600, 90 and 500, 100 and 400,110 and 300, or 120 and 200 nucleotides in length (with or withoutheterologous sequences). In embodiment the nucleic acid is between 50and 1,000, 50 and 900, 50 and 800, 50 and 700, 50 and 600, 50 and 500,50 and 400, 50 and 300, or 50 and 200 nucleotides in length (with orwithout heterologous sequences). In embodiments the nucleic acid is ofsufficient length to allow sequencing (e.g., by chemical sequencing orby determining a difference in T_(m) between mutant and referencepreparations) but is optionally less than 100, 200, 300, 400, or 500nucleotides in length (with or without heterologous sequences).

Such preparations can be used to sequence nucleic acid from a sample,e.g., a tumor sample. In an embodiment the purified preparation isprovided by in situ amplification of a nucleic acid provided on asubstrate. In embodiments the purified preparation is spatially distinctfrom other nucleic acids, e.g., other amplified nucleic acids, on asubstrate.

In an embodiment the purified or isolated preparation of nucleic acid isderived from a tumor of a type described herein, e.g., an ER⁺ cancer, ora breast cancer, e.g., a breast cancer, e.g., a breast cancer that isone, two or all of ER⁺, malignant, or resistant to treatment, e.g.,treatment with a first or second line therapy, e.g., a SERM, e.g., aSERM described herein, e.g., tamoxifen. In an embodiment the ESR1nucleic acid is derived from a breast cancer that is ER⁺ and resistantto treatment, e.g., treatment with a first or second line therapy, e.g.,a SERM, e.g., a SERM described herein, e.g., tamoxifen.

Such preparations can be used to determine if a sample comprises mutantsequence.

In another aspect, the invention features, a method of determining thesequence of an interrogation position for a mutation described herein,comprising:

providing a purified or isolated preparations of an ER⁺ tumor cellnucleic acid or ESR1 nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA,or RNA, containing an interrogation position described herein,

sequencing, by a method that breaks or forms a chemical bond, e.g., acovalent or non-covalent chemical bond, e.g., in a detection reagent ora target sequence, the nucleic acid so as to determine the identity ofthe nucleotide at an interrogation position. The method allowsdetermining if a mutation described herein is present.

In an embodiment, sequencing comprises contacting the ESR1 nucleic acidwith a detection reagent described herein.

In an embodiment, sequencing comprises determining a physical property,e.g., stability of a duplex form of the ESR1 nucleic acid, e.g., T_(m),that can distinguish mutant from reference sequence.

In an embodiment, the ESR1 nucleic acid is derived from a tumor of atype described herein, e.g., a breast cancer, e.g., a breast cancer,e.g., a breast cancer that is one, two or all of ER⁺, malignant, orresistant to treatment, e.g., treatment with a first or second linetherapy, e.g., a SERM, e.g., a SERM described herein, e.g., tamoxifen.In an embodiment the ESR1 nucleic acid is derived from an ER⁺ cancer,e.g., a breast cancer that is ER⁺ and resistant to treatment, e.g.,treatment with a first or second line therapy, e.g., a SERM, e.g., aSERM described herein, e.g., tamoxifen.

Reaction Mixtures and Devices

In another aspect, the invention features, purified or isolatedpreparations of a ESR1 nucleic acid, e.g., DNA, e.g., genomic DNA orcDNA, or RNA, containing an interrogation position described herein,useful for determining if a mutation disclosed herein is present,disposed in sequencing device, or a sample holder for use in such adevice. In an embodiment the ESR1 nucleic acid is derived from a tumorof a type described herein, e.g., a breast cancer, e.g., a breast cancerthat is one, two or all of ER⁺, malignant, or resistant to treatment,e.g., treatment with a first or second line therapy, e.g., a SERM, e.g.,a SERM described herein, e.g., tamoxifen. In an embodiment the ESR1nucleic acid is derived from an ER⁺ cancer, e.g., a breast cancer thatis ER⁺ and resistant to treatment, e.g., treatment with a first orsecond line therapy, e.g., a SERM, e.g., a SERM described herein, e.g.,tamoxifen.

In another aspect, the invention features, purified or isolatedpreparations of a ESR1 nucleic acid, e.g., DNA, e.g., genomic DNA orcDNA, or RNA, containing an interrogation position described herein,useful for determining if a mutation disclosed herein is present,disposed in a device for determining a physical or chemical property,e.g., stability of a duplex, e.g., T_(m) or a sample holder for use insuch a device. In an embodiment the device is a calorimeter. In anembodiment the ESR1 nucleic acid is derived from a tumor of a typedescribed herein, e.g., an ER⁺ cancer or a breast cancer, e.g., a breastcancer that is one, two or all of ER⁺, malignant, or resistant totreatment, e.g., treatment with a first or second line therapy, e.g., aSERM, e.g., a SERM described herein, e.g., tamoxifen. In an embodimentthe ESR1 nucleic acid is derived from a breast cancer that is ER⁺ andresistant to treatment, e.g., treatment with a first or second linetherapy, e.g., a SERM, e.g., a SERM described herein, e.g., tamoxifen.

The detection reagents described herein can be used to determine if amutation described herein is present in a sample. In embodiments thesample comprises a nucleic acid that is derived from a tumor cell. Thetumor cell can be from a tumor sample, e.g., a biopsy taken from thetumor, or from circulating tumor cells, e.g., from peripheral blood. Inan embodiment the ESR1 nucleic acid is derived from a tumor of a typedescribed herein, e.g., an ER⁺ cancer or a breast cancer, e.g., a breastcancer that is one, two or all of ER⁺, malignant, or resistant totreatment, e.g., treatment with a first or second line therapy, e.g., aSERM, e.g., a SERM described herein, e.g., tamoxifen. In an embodimentthe ESR1 nucleic acid is derived from a breast cancer that is ER⁺ andresistant to treatment, e.g., treatment with a first or second linetherapy, e.g., a SERM, e.g., a SERM described herein, e.g., tamoxifen.

Accordingly, in one aspect, the invention features a method of making areaction mixture comprising:

combining a detection reagent, or purified or isolated preparationthereof, described herein with a target nucleic acid derived from atumor cell which comprises a sequence having an interrogation positionfor a mutation described herein.

In another aspect, the invention features, a reaction mixturecomprising:

a detection reagent, or purified or isolated preparation thereof,described herein; and

a target nucleic acid derived from a tumor cell which comprises asequence having an interrogation position for a mutation describedherein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture:

the detection reagent comprises a nucleic acid, e.g., a DNA, RNA ormixed DNA/RNA, molecule which is complementary with a nucleic acidsequence on a target nucleic acid (the detection reagent binding site)wherein the detection reagent binding site is disposed in relationshipto the interrogation position such that binding of the detection reagentto the detection reagent binding site allows differentiation of mutantand reference sequences for a mutant described herein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture:

the tumor is a tumor described herein, e.g., an ER⁺ cancer or a breastcancer, e.g., a breast cancer that is one, two or all of ER⁺, malignant,or resistant to treatment, e.g., treatment with a first or second linetherapy, e.g., a SERM, e.g., a SERM described herein, e.g., tamoxifen.In an embodiment the ESR1 nucleic acid is derived from a breast cancerthat is ER⁺ and resistant to treatment, e.g., treatment with a first orsecond line therapy, e.g., a SERM, e.g., a SERM described herein, e.g.,tamoxifen.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the mutation is a mutation described herein,including: a 6 nucleotide deletion of nucleotides 1046-1051 (TGGCAG)according to SEQ ID NO:1 or other deletion, e.g., an in-frame deletion,that includes one or more of nucleotides 1046-1051; a mutation at aposition identified as mutated in Table 3, e.g., any of the mutationsdescribed in Table 3; or, an associated mutation.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the mutation is at position 537 or 538 of the aminoacid sequence of SEQ ID NO:2 (FIGS. 2A-2B).

In an embodiment of the reaction mixture, or the method of making thereaction mixture the mutation is selected from:

a missense mutation at amino acid 537 of SEQ ID NO:2;

a missense mutation at nucleotide 1610 of SEQ ID NO:

a tyrosine to asparagine substitution at position 537 (a Y537N);

a tyrosine to cysteine substitution at position 537 (a Y537C);

a T to A replacement at nucleotide 1609; or

an A to G replacement at nucleotide 1610.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the mutation is selected from:

-   -   a missense mutation at amino acid 538 of SEQ ID NO:2;

a missense mutation at nucleotide 1613 of SEQ ID NO:1;

an aspartate to glycine substitution at position 538 (a D538G), of SEQID NO:2; or

an A to G replacement at nucleotide 1613, of SEQ ID NO:1.

A mutation described herein, can be distinguished from a reference,e.g., a non-mutant or wildtype sequence, by reaction with an enzyme thatreacts differentially with the mutation and the reference. E.g., theycan be distinguished by cleavage with a restriction enzyme that hasdiffering activity for the mutant and reference sequences. E.g., theinvention includes a method of contacting a nucleic acid comprising amutation described herein with a such an enzyme and determining if aproduct of that cleavage which can distinguish mutant form referencesequence is present.

In one aspect, the inventions provides, a purified preparation of arestriction enzyme cleavage product which can distinguish between mutantand reference sequence, wherein one end of the cleavage product isdefined by an enzyme that cleaves differentially between mutant andreference sequence. In an embodiment the cleavage product includes theinterrogation position.

Protein-Based Detection Reagents, Methods, Reaction Mixtures and Devices

A mutant protein described herein can be distinguished from a reference,e.g., a non-mutant or wildtype protein, by reaction with a reagent,e.g., a substrate, e.g, a substrate for phosphorylation or other ESR1activity, or an antibody, that reacts differentially with the mutant andreference protein. In one aspect, the invention includes a method ofcontacting a sample comprising a mutant protein described herein with asuch reagent and determining if the mutant protein is present in thesample.

In another embodiment, the invention features, an antibody that candistinguish a mutant protein described herein, e.g., a mutant proteincorresponding to a mutation in Table 3 or an associated mutation from areference, e.g., a non-mutant or wildtype protein.

Accordingly, in one aspect, the invention features a method of making areaction mixture comprising:

combining a detection reagent, or purified or isolated preparationthereof, e.g., a substrate, e.g., a substrate for phosphorylation orother activity, or an antibody, described herein with a target ESR1protein derived from a tumor cell which comprises a sequence having aninterrogation position for a mutation described herein.

In another aspect, the invention features, a reaction mixturecomprising:

a detection reagent, or purified or isolated preparation thereof, e.g.,a substrate, e.g., a substrate for phosphorylation or other activity, oran antibody, described herein; and

a target ESR1 protein derived from a tumor cell which comprises asequence having an interrogation position for a mutation describedherein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture:

the detection reagent comprises an antibody specific for a mutant ESR1protein described herein.

In an embodiment of the reaction mixture, or the method of making thereaction mixture:

the tumor is a tumor described herein, e.g., an ER⁺ cancer or a breastcancer, e.g., a breast cancer that is one, two or all of ER⁺, malignant,or resistant to treatment, e.g., treatment with a first or second linetherapy, e.g., a SERM, e.g., a SERM described herein, e.g., tamoxifen.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the mutation is a mutation described herein,including: a 6 nucleotide deletion of nucleotides 1046-1051 (TGGCAG)according to SEQ ID NO:1 or other deletion, e.g., an in-frame deletion,that includes one or more of nucleotides 1046-1051; a mutation at aposition identified as mutated in Table 3, e.g., any of the mutationsdescribed in Table 3; or, an associated mutation.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the mutation is at position 537 or 538 of the aminoacid sequence of SEQ ID NO:2 (FIGS. 2A-2B).

In an embodiment of the reaction mixture, or the method of making thereaction mixture the mutation is selected from:

a missense mutation at amino acid 537 of SEQ ID NO:2;

a missense mutation at nucleotide 1610 of SEQ ID NO:

a tyrosine to asparagine substitution at position 537 (a Y537N);

a tyrosine to cysteine substitution at position 537 (a Y537C);

a T to A replacement at nucleotide 1609; or

an A to G replacement at nucleotide 1610.

In an embodiment of the reaction mixture, or the method of making thereaction mixture: the mutation is selected from:

a missense mutation at amino acid 538 of SEQ ID NO:2;

a missense mutation at nucleotide 1613 of SEQ ID NO:1;

an aspartate to glycine substitution at position 538 (a D538G), of SEQID NO:2; or

an A to G replacement at nucleotide 1613, of SEQ ID NO:1.

Kits

In another aspect, the invention features a kit comprising a detectionreagent described herein.

Method of Evaluating a Cancer or a Subject

In another aspect, the invention features a method of evaluating asubject (e.g., a patient), e.g., for risk of having or developing acancer, e.g., a breast cancer, a colorectal cancer, a lung cancer, orprostate cancer. The method includes: acquiring information or knowledgeof the presence of a mutant ESR1 in a subject (e.g., acquiring genotypeinformation of the subject that identifies a mutant ESR1 as beingpresent in the subject); acquiring a sequence for a nucleic acidmolecule identified herein (e.g., a nucleic acid molecule that includesa mutant ESR1 sequence); or detecting the presence of a mutant ESR1nucleic acid or polypeptide in the subject), wherein the presence of themutant ESR1 is positively correlated with increased risk for, or having,a cancer associated with the mutant ESR1.

The method can further include the step(s) of identifying (e.g.,evaluating, diagnosing, screening, and/or selecting) the subject asbeing positively correlated with increased risk for, or having, a cancerassociated with the mutant ESR1. In one embodiment, the subject isidentified or selected as likely or unlikely to respond to a treatment,e.g., a SERM or an aromatase inhibitor treatment as described herein.

The method can further include treating the subject with an anti-canceragent, e.g., a SERM, an aromatase inhibitor, a SERD, an estrogenreceptor modulator, or an mTOR pathway inhibitor, as described herein.

The method can further include acquiring, e.g., directly or indirectly,a sample from a patient and evaluating the sample for the presence of amutant ESR1 as described herein.

In one embodiment, a subject having a cancer, e.g., a breast cancer, isevaluated for the presence of a mutant ESR1, such as a mutation in theligand binding domain of ESR1. In one embodiment, the subject has anER-positive cancer, e.g., an ER-positive breast cancer (detected by,e.g., IHC). A subject identified as not having a mutant ESR1 can beadministered a SERM, and the patient can be monitored at regularintervals, e.g., monthly, or once every three, six, 8 or 12 months, orat shorter or longer intervals, for the presence of a mutant ESR1. Ifduring the course of SERM therapy, the subject is found to carry an ESR1mutation, the subject can stop receiving SERM therapy, or can beadministered a decreased dose of the SERM therapy. If the subject is apost-menopausal female patient, the patient may begin treatment with anaromatase therapy. If the subject is a pre-menopausal female patient,the patient may begin treatment with an alternative estrogen receptorblocker, or may receive an oophorectomy. In one embodiment, the subjectcontinues to receive the SERM therapy. A patient who carries a mutationin the ligand binding domain of ESR1, such as a mutation describedherein, and who continues to receive the SERM therapy (at the same or ata decreased dosage), may undergo increased monitoring for a worsening incancer symptoms. In one embodiment, the dose of SERM is decreased overtime.

In certain embodiments, the subject is a patient or patient populationthat has participated in a clinical trial. In one embodiment, thesubject has participated in a clinical trial for evaluating an estrogeninhibitor (e.g., a SERM or an aromatase inhibitor). In one embodiment,the estrogen inhibitor was not a SERM. In one embodiment, the clinicaltrial is discontinued or terminated. In one embodiment, the subjectresponded favorably to the clinical trial, e.g., experienced animprovement in at least one symptom of a cancer (e.g., decreased intumor size, rate of tumor growth, increased survival). In otherembodiments, the subject did not respond in a detectable way to theclinical trial. In one embodiment, the presence of a mutant ESR1, e.g.,an ESR1 comprising a mutation in the ligand binding domain identifiesthe patient as being a candidate to receive treatment with an agentother than a SERM. In another embodiment, the presence of the mutantESR1 identifies the patient as being a candidate to receive treatmentwith an aromatase inhibitor or fulvestrant, or an alternative estrogenreceptor blocking agent.

In a related aspect, a method of evaluating a patient or a patientpopulation is provided. The method includes: identifying, selecting, orobtaining information or knowledge that the patient or patientpopulation has participated in a clinical trial;

acquiring information or knowledge of the presence of a mutant ESR1 inthe patient or patient population (e.g., acquiring genotype informationof the subject that identifies a mutant ESR1 as being present in thesubject); acquiring a sequence for a nucleic acid molecule identifiedherein (e.g., a nucleic acid molecule that includes a mutant ESR1sequence); or detecting the presence of a mutant ESR1 nucleic acid orpolypeptide in the subject), wherein the presence of the mutant ESR1identifies the patient or patient population as having an increased riskfor, or having, a cancer associated with the mutant ESR1.

In certain embodiments, the subject is a patient or patient populationthat has participated in a clinical trial. In one embodiment, thesubject has participated in a clinical trial for evaluating a SERM or anaromatase inhibitor. In one embodiment, the clinical trial isdiscontinued or terminated. In one embodiment, the subject respondedfavorably to the clinical trial, e.g., experience an improvement in atleast one symptom of a cancer (e.g., decreased in tumor size, rate oftumor growth, increased survival). In other embodiments, the subject didnot respond in a detectable way to the clinical trial.

In embodiments, the method further includes treating the subject with aSERM or an aromatase inhibitor, e.g., an aromatase inhibitor asdescribed herein.

Reporting

Methods described herein can include providing a report, such as, inelectronic, web-based, or paper form, to the patient or to anotherperson or entity, e.g., a caregiver, e.g., a physician, e.g., anoncologist, a hospital, clinic, third-party payor, insurance company orgovernment office. The report can include output from the method, e.g.,the identification of nucleotide values, the indication of presence orabsence of a mutant ESR1 as described herein, or sequence. In oneembodiment, a report is generated, such as in paper or electronic form,which identifies the presence or absence of an alteration describedherein, and optionally includes an identifier for the patient from whichthe sequence was obtained. In one embodiment, the report is in web-basedform.

The report can also include information on the role of a sequence, e.g.,a mutant ESR1 as described herein, or wild-type sequence, in disease.Such information can include information on prognosis, resistance, orpotential or suggested therapeutic options. The report can includeinformation on the likely effectiveness of a therapeutic option, theacceptability of a therapeutic option, or the advisability of applyingthe therapeutic option to a patient, e.g., a patient having a sequence,alteration or mutation identified in the test, and in embodiments,identified in the report. For example, the report can includeinformation, or a recommendation on, the administration of a drug, e.g.,the administration at a preselected dosage or in a preselected treatmentregimen, e.g., in combination with other drugs, to the patient. In anembodiment, not all mutations identified in the method are identified inthe report. For example, the report can be limited to mutations in geneshaving a preselected level of correlation with the occurrence,prognosis, stage, or susceptibility of the cancer to treatment, e.g.,with a preselected therapeutic option. The report can be delivered,e.g., to an entity described herein, within 7, 14, or 21 days fromreceipt of the sample by the entity practicing the method.

In another aspect, the invention features a method for generating areport, e.g., a personalized cancer treatment report, by obtaining asample, e.g., a tumor sample, from a subject, detecting a mutant ESR1 asdescribed herein in the sample, and selecting a treatment based on themutation identified. In one embodiment, a report is generated thatannotates the selected treatment, or that lists, e.g., in order ofpreference, two or more treatment options based on the mutationidentified. In another embodiment, the subject, e.g., a patient, isfurther administered the selected method of treatment.

In one embodiment, a report is generated to memorialize each time apatient is tested for an ESR1 mutation. For example, a patient who isdetermined not to have an ESR1 mutation can be administered a SERM totreat a cancer, such as a breast cancer. The patient can be reevaluatedat intervals, such as every month, every two months, every six months orevery year, or more or less frequently, to monitor the patient for thedevelopment of a mutation in ESR1, e.g., in the ligand binding domain ofESR1. If the patient is subsequently determined to have a mutant ESR1,administration of the SERM to the patient can be stopped, and thepatient can be administered an anti-cancer agent that is not a SERM. Inone embodiment, the patient is a post-menopausal female patient whoadministered an aromatase inhibitor or fulvestrant, or an mTOR pathwayinhibitor. In another embodiment, the patient is a pre-menopausal femalepatient who is administered an alternative estrogen receptor blocker, oran oophorectomy. In yet another embodiment, the patient continues toreceive treatment with the SERM, or receives a lower dose of the SERM,and optionally the patient is monitored more frequently for a worseningof cancer symptoms. The report can record at least the treatment historyof the patient and the corresponding result of each test to assay for anESR1 mutation in the patient.

Systems or Devices

In another aspect, the invention features a system or a device (e.g., asequencing device) for producing a report, e.g., a genotype report. Thesystem or device can include a component for containing a sample (e.g.,a tumor nucleic acid or polypeptide); a detection component capable ofidentifying the presence or absence of a mutant ESR1 as describedherein; and a means for outputting a report, e.g., a report as describedherein.

In one embodiment, the component for containing a tumor sample isconfigured in a way to contain or hold the sample, e.g., a tumor nucleicacid or polypeptide sample.

In another embodiment, the detection component produces and/or analyzesa signal according to the presence or absence of the mutant ESR1 in thesample.

In another embodiment, the means for outputting a report provides asystem for annotating the association of the detected mutant ESR1 to thesample. The report can include, e.g., the identification of nucleotidevalues, the indication of presence or absence of a mutant ESR1 asdescribed herein, or sequence. In one embodiment, a report is generated,such as in paper or electronic form, which identifies the presence orabsence of an alteration described herein, and optionally includes anidentifier for the patient from which the sequence was obtained.

The report can also include information on the role of a sequence, e.g.,a mutant ESR1 as described herein, or wild-type sequence, in disease.Such information can include information on prognosis, resistance, orpotential or suggested therapeutic options. The report can includeinformation on the likely effectiveness of a therapeutic option, theacceptability of a therapeutic option, or the advisability of applyingthe therapeutic option to a patient, e.g., a patient having a sequence,alteration or mutation identified in the test, and in embodiments,identified in the report.

Methods of Reducing a Mutant ESR1 Activity

In another aspect, the invention features a method of reducing anactivity of a mutant ESR1. The method includes administering an estrogeninhibitor, such as a SERM, aromatase or fulvestrant. In one embodiment,the estrogen inhibitor is not a SERM.

An “estrogen inhibitor” as used herein is any molecule that interfereswith the estrogen signaling pathway, including SERMs and estrogenmimetics, which compete for binding with estrogen in the ligand bindingdomain of ESR1, and aromatase inhibitors, which interfere with theproduction of estrogen. An estrogen inhibitor can be an estrogenmimetic, which can interact with estrogen receptor to have apro-estrogenic (estrogenic) or an anti-estrogenic effect. Apro-estrogenic, or estrogenic, response exaggerates or enhances theeffect of an estrogen, such as by increasing transcription of gene undercontrol of an estrogen response element. An anti-estrogenic responseinhibits an estrogen-dependent activity, e.g., transcription of a geneunder control of an estrogen response element is decreased in responseto a molecule that has an anti-estrogenic effect.

An estrogen inhibitor can be a small molecule compound (e.g., a hormone,such as synthetic hormone), a nucleic acid (e.g., antisense, siRNA,aptamer, ribozymes, microRNA), or a protein, such as an antibodymolecule (e.g., a full antibody or antigen binding fragment thereof thatbinds to mutant ESR1). The candidate agent can be obtained from alibrary (e.g., a commercial library of estrogen inhibitors, such asSERMs or aromatase inhibitors) or can be rationally designed (e.g.,based on the ligand binding domain).

In one embodiment, the estrogen inhibitor is administered based on adetermination that a mutant ESR1 is present in a subject, e.g., based onits presence in a subject's sample. Thus, treatment can be combined witha mutant ESR1 detection or evaluation method, e.g., as described herein,or administered in response to a determination made by a mutant ESR1detection or evaluation method, e.g., as described herein. In certainembodiments, the estrogen inhibitor is administered responsive toacquiring knowledge or information of the presence of the mutant ESR1 ina subject. In one embodiment, the estrogen inhibitor is administeredresponsive to acquiring knowledge or information on the subject'sgenotype, e.g., acquiring knowledge or information that the patient'sgenotype has a mutation in the ESR1 gene. In other embodiments, theestrogen inhibitor is administered responsive to receiving acommunication (e.g., a report) of the presence of the mutant ESR1 in asubject (e.g., a subject's sample). In yet other embodiments, theestrogen inhibitor is administered responsive to information obtainedfrom a collaboration with another party that identifies the presence ofthe mutant ESR1 in a subject (e.g., a subject's sample). In otherembodiments, the estrogen inhibitor is administered responsive to adetermination that the mutant ESR1 is present in a subject. In oneembodiment, the determination of the presence of the mutant ESR1 iscarried out using one or more of the methods, e.g., the sequencingmethods, described herein. In other embodiments, the determination ofthe presence of the mutant ESR1 includes receiving information on thesubject's mutant ESR1 genotype, e.g., from another party or source.

The methods can, optionally, further include the step(s) of identifying(e.g., evaluating, diagnosing, screening, and/or selecting) a subject atrisk of having, or having, a mutant ESR1. In one embodiment, the methodfurther includes one or more of: acquiring knowledge or information ofthe presence of the mutant ESR1 in a subject (e.g., a subject's sample);acquiring knowledge or information on the subject's genotype, e.g.,acquiring knowledge or information that the patient's genotype has amutant ESR1; receiving a communication (e.g., a report) of the presenceof the mutant ESR1 in a subject (e.g., a subject's sample); orcollaborating with another party that identifies the presence of themutant ESR1 in a subject.

In one embodiment, the subject treated has a mutant ESR1; e.g., thesubject has a tumor or cancer harboring a mutant ESR1, such as amutation in the ligand binding domain. In other embodiments, the subjecthas been previously identified as having a mutant ESR1.

In one embodiment, a SERM causes an estrogenic effect on the mutant ESR.In another embodiment, the subject is administered an anti-cancer agentthat is not a SERM. For example, the subject is a post-menopausal femalepatient administered an aromatase inhibitor or fulvestrant. In anotherembodiment, the aromatase inhibitor or fulvestrant is administeredresponsive to the determination of the presence of the mutant ESR1 in atumor sample from the subject.

In one embodiment, the subject was previously tested for the presence ofa mutant ESR1 and a mutation in the ligand binding domain of ESR1 wasnot detected. The subject was further administered a SERM based on theknowledge that a mutation in the ligand binding domain of ESR1 was notdetected. In one embodiment, the subject was further tested at intervals(e.g., monthly, or every 3, 4, 6 months, or every year, or more or lessfrequently) for the presence of a mutant ESR1, and where a mutation inthe ligand binding domain of ESR1 was not detected, the subjectcontinued treatment with a SERM based on the knowledge that a mutationin the ligand binding domain of ESR1 was not detected. In oneembodiment, the subject was further tested at intervals for the presenceof a mutant ESR1, and where a mutation in the ligand binding domain ofESR1 was detected, the subject stopped treatment with the SERM based onthe knowledge that a mutation in the ligand binding domain of ESR1 wasdetected. In another embodiment, the subject further began treatmentwith an anti-cancer agent that was not a SERM. For example, in oneembodiment, the subject was a post-menopausal female patient, and thepatient began treatment with an aromatase inhibitor or fulvestrant. Inanother embodiment, the subject was a pre-menopausal female patient, andthe patient was administered an alternative estrogen receptor blocker oran oophorectomy. In another embodiment, the subject maintained treatmentwith the SERM, or was administered a lower dose of the SERM based on theknowledge that a mutation in the ligand binding domain of ESR1 wasdetected, and in yet another embodiment, the patient was administered acombination of a lower dose of SERM and a second anti-cancer agent, suchas an aromatase inhibitor or fulvestrant. In one embodiment, where thepatient was continued to receive treatment with the SERM, the patientreceived increased monitoring for a worsening of cancer symptoms, suchas described herein.

In yet other embodiments, the subject has been previously identified asbeing likely or unlikely to respond to treatment with a SERM, e.g., asubject that has previously participated in a clinical trial. In otherembodiments, the subject has been previously identified as being likelyor unlikely to respond to treatment with a SERM, based on the presenceof the mutant ESR1.

Methods of reducing an activity of a mutant ESR1 include contacting themutant ESR1, or a mutant ESR1-expressing cell, with an agent thatinhibits an activity or expression of mutant ESR1, e.g., an agent thatblocks the transcriptional activation activity of ESR1. In oneembodiment, the contacting step can be effected in vitro, e.g., in acell lysate or in a reconstituted system. Alternatively, the method canbe performed on cells in culture, e.g., in vitro or ex vivo. In otherembodiments, the method can be performed on mutant ESR1-expressing cellspresent in a subject, e.g., as part of an in vivo (e.g., therapeutic orprophylactic) protocol. In an embodiment, the method is practiced on ananimal subject (e.g., an in vivo animal model). In certain embodiments,the mutant ESR1 is a nucleic acid molecule or a polypeptide as describedherein.

In a related aspect, a method of inhibiting, reducing, or treating ahyperproliferative disorder, e.g., a cancer, in a subject is provided.The method includes administering to the subject a preselectedtherapeutic agent, e.g., an anti-cancer agent (e.g., estrogen inhibitor,such as an aromatase inhibitor or a SERM), as a single agent, or incombination, in an amount sufficient to reduce, inhibit or treat theactivity or expression of mutant ESR1 (e.g., a mutant ESR1 describedherein), thereby inhibiting, reducing, or treating thehyperproliferative disorder in the subject. “Treatment” as used hereinincludes, but is not limited to, inhibiting tumor growth, reducing tumormass, reducing size or number of metastatic lesions, inhibiting thedevelopment of new metastatic lesions, prolonged survival, prolongedprogression-free survival, prolonged time to progression, and/orenhanced quality of life.

In one embodiment, the subject is a mammal, e.g., a human. In oneembodiment, the subject has, or is at risk of having a cancer at anystage of disease. In other embodiments, the subject is a patient, e.g.,a cancer patient.

In other embodiments, the subject treated is a cancer patient who hasparticipated in a clinical trial. For example, the subject participatedin a clinical trial that evaluated a SERM. In one embodiment, the cancerpatient responded to the SERM evaluated.

In certain embodiments, the cancer is a solid tumor, a soft tissuetumor, or a metastatic lesion. In one embodiment, the cancer is chosenfrom a breast cancer, prostate cancer, ovarian cancer, endometrialcancer, colon cancer, or a combination thereof. In one embodiment, thecancer is a metastatic cancer.

In one embodiment, the anti-cancer agent is an estrogen inhibitor, suchas an aromatase inhibitor. For example, the aromatase inhibitor isaminoglutethimide, testolactone (Teslac®), anastrozole (Arimidex®),letrozole (Femara®), exemestane (Aromasin®), vorozole (Rivizor),formestane (Lentaron®), fadrozole (Afema); 4-hydroxyandrostenedione,1,4,6-androstatrien-3,17-dione (ATD), and 4-Androstene-3,6,17-trione(“6-OXO”).

In other embodiments, the anti-cancer agent is a mutant ESR1 antagonistthat inhibits the expression of a nucleic acid encoding mutant ESR1.Examples of such mutant ESR1 antagonists include nucleic acid molecules,for example, antisense molecules, ribozymes, RNAi, triple helixmolecules that hybridize to a nucleic acid encoding a mutant ESR1, or atranscription regulatory region, and blocks or reduces mRNA expressionof mutant ESR1.

In other embodiments, the estrogen inhibitor is administered incombination with a second therapeutic agent or a different therapeuticmodality, e.g., anti-cancer agents, and/or in combination with surgicaland/or radiation procedures. For example, the second therapeutic agentcan be a cytotoxic or a cytostatic agent. Exemplary cytotoxic agentsinclude antimicrotubule agents, topoisomerase inhibitors, or taxanes,anti-metabolites, mitotic inhibitors, alkylating agents, intercalatingagents, agents capable of interfering with a signal transductionpathway, agents that promote apoptosis and radiation. In yet otherembodiments, the methods can be used in combination with immunodulatoryagents, e.g., IL-1, 2, 4, 6, or 12, or interferon α or γ, or immune cellgrowth factors such as GM-CSF.

Screening Methods

In another aspect, the invention features a method, or assay, forscreening for agents that modulate, e.g., inhibit, the expression oractivity of a mutant ESR1, e.g., a mutant ESR1 as described herein. Themethod includes contacting a mutant ESR1 nucleic acid or polypeptide, ora cell expressing a mutant ESR1 nucleic acid or polypeptide, with acandidate agent; and detecting a change in a parameter associated with amutant ESR1, e.g., a change in the expression or an activity of themutant ESR1. The method can, optionally, include comparing the treatedparameter to a reference value, e.g., a control sample (e.g., comparinga parameter obtained from a sample with the candidate agent to aparameter obtained from a sample without the candidate agent). In oneembodiment, if a decrease in expression or activity of the mutant ESR1is detected, the candidate agent is identified as an inhibitor. Inanother embodiment, if an increase in expression or activity of themutant ESR1 is detected, the candidate agent is identified as anactivator. In certain embodiments, the mutant ESR1 is a nucleic acidmolecule or a polypeptide as described herein.

In one embodiment, the contacting step is effected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is effected in a cell in culture, e.g.,a cell expressing a mutant ESR1 (e.g., a mammalian cell, a tumor cell orcell line, a recombinant cell). In yet other embodiments, the contactingstep is effected in a cell in vivo (a mutant ESR1-expressing cellpresent in a subject, e.g., an animal subject (e.g., an in vivo animalmodel).

Exemplary parameters evaluated include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to a mutant ESR1 polypeptide, e.g., an ESR1 carrying a mutation inthe ligand binding domain; a binding competition between a known ligand(e.g., estrogen or an estrogen mimic) and the candidate agent to amutant ESR1 polypeptide;

(ii) a change in transcriptional activation activity, e.g., expressionlevels of a reporter gene under control of an estrogen response element;or a change in DNA binding activity, such as binding at an estrogenresponse element; In certain embodiments, a change in DNA bindingactivity is detected by gel shift analysis, or by assaying expressionfrom a reporter gene;

(iii) a change in an activity of a cell containing a mutant ESR1 (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology or tumorigenicity of the cell;

(iv) a change in a tumor present in an animal subject, e.g., size,appearance, proliferation, of the tumor; or

(v) a change in the level, e.g., expression level, of a mutant ESR1polypeptide or nucleic acid molecule.

In one embodiment, a change in a cell free assay in the presence of acandidate agent is evaluated. For example, an activity of a mutant ESR1,or interaction of a mutant ESR1 with a ligand can be detected. In oneembodiment, a mutant ESR1 polypeptide is contacted with a ligand, e.g.,in solution, and a candidate agent is monitored for an ability tomodulate, e.g., inhibit, an interaction, e.g., binding, between themutant ESR1 polypeptide and the ligand (e.g., estrogen or an estrogenmimic).

In other embodiments, a change in an activity of a cell is detected in acell in culture, e.g., a cell expressing a mutant ESR1 (e.g., amammalian cell, a tumor cell or cell line, a recombinant cell). In oneembodiment, the cell is a recombinant cell that is modified to express amutant ESR1 nucleic acid, e.g., is a recombinant cell transfected with amutant ESR1 nucleic acid. The transfected cell can show a change inresponse to the expressed mutant ESR1, e.g., increased proliferation,changes in morphology, increased tumorigenicity, and/or an acquiredtransformed phenotype. A change in any of the activities of the cell,e.g., the recombinant cell, in the presence of the candidate agent canbe detected. For example, a decrease in one or more of: proliferation,tumorigenicity, transformed morphology, in the presence of the candidateagent can be indicative of an inhibitor of a mutant ESR1. In otherembodiments, a change in binding activity or phosphorylation asdescribed herein is detected.

In yet other embodiments, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,the animal model is a tumor containing animal or a xenograft comprisingcells expressing a mutant ESR1 (e.g., tumorigenic cells expressing amutant ESR1). The candidate agent can be administered to the animalsubject and a change in the tumor is detected. In one embodiment, thechange in the tumor includes one or more of a change in tumor growth,tumor size, tumor burden, and survival. A decrease in one or more oftumor growth, tumor size, tumor burden, or an increased survival isindicative that the candidate agent is an inhibitor.

In other embodiments, a change in expression of a mutant ESR1 can bemonitored by detecting the nucleic acid or protein levels, e.g., usingthe methods described herein.

In certain embodiments, the screening methods described herein can berepeated and/or combined. In one embodiment, a candidate agent that isevaluated in a cell-free or cell-based described herein can be furthertested in an animal subject.

In one embodiment, the candidate agent is a small molecule compound,e.g., an estrogen inhibitor, such as a SERM or an aromatase inhibitor, anucleic acid (e.g., antisense, siRNA, aptamer, ribozymes, microRNA), anantibody molecule (e.g., a full antibody or antigen binding fragmentthereof that binds to mutant ESR1). The candidate agent can be obtainedfrom a library (e.g., a commercial library of estrogen inhibitors, suchas SERMs or aromatase inhibitors) or rationally designed (e.g., based onthe ligand binding domain).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, and theexample are illustrative only and not intended to be limiting.

The details of one or more embodiments featured in the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages featured in the invention will beapparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of the structure of the ESR1 protein. TheDNA-binding domain (DBD), hinge region and ligand binding domain areindicated. The Activation Function (AF) domains and exon boundaries arealso indicated. Exons are numbered according to the numbering in RefSeq.NM_001122742 (Oct. 2, 2011).

FIGS. 2A and 2B are the cDNA (SEQ ID NO:1) and amino acid (SEQ ID NO:2)sequence alignment for wildtype ESR1 (RefSeq. NM_000125.3, Oct. 2,2011). The deleted nucleotides at positions 1046-1051 and amino acids atpositions 349-351 in the novel mutation are underlined and indicated inbold type in FIG. 2B. Underlined codons and amino acids indicate thesites of point mutations identified in Table 3.

FIG. 3 is a snapshot of the sequencing reads illustrating that there isa 6 nucleotide deletion (TTGCAG) in the ESR1 gene, which causesreplacement of amino acids LAD at positions 349-351 of the ESR1 protein,with H (See FIG. 2B). FIG. 3 discloses SEQ ID NOs: 7-11, respectively,in order of appearance.

FIGS. 4A-4B are the cDNA (SEQ ID NO:3) and amino acid (SEQ ID NO:4)sequence alignments for the novel mutation in ESR1. The fusion junctioncreated by the deletion, and the subsequent substation of H for thedeleted amino acids LAD are indicated in bold type and by underlining inFIG. 4B. A bar (“|”) marks the fusion junction.

DETAILED DESCRIPTION

The invention is based, at least in part, on the discovery of a novelmutation in the ligand binding domain of the estrogen receptor a gene(ESR1) and its association with cancer, e.g., breast cancer. In oneembodiment, Applicants have discovered a novel deletion on chromosome 6that results in deletion of 6 nucleotides and subsequently thesubstitution of a histidine for a leucine-adenine-aspartic acid deletionin the ligand binding domain of ESR1.

The ESR1 gene is associated with cancerous phenotypes, including breastcancer, ovarian cancer, endometrial cancer, prostate cancer and coloncancer, among others. For example, point mutations in ESR1 associatedwith breast cancer include the following alterations: S47T, N69K, andA86V, each individually in the AF-1 domain; L296P, and K303R in theregion between the hinge and AF-2 domains, and E352V, M396V, 437stop,K531E, and Y537N in the hormone binding domain in AF-2 (Herynk andFuqua, Endocrine Reviews 25:869-898, 2004). A breast cancer patientcarrying the E352V mutation responded to adjuvant tamoxifen therapy(Id.). A naturally occurring mutation in the ligand binding domain wasdiscovered in the tamoxifen-resistant MCF7/MT2 xenografted tumor line.The D351Y mutation was the primary form of ESR1 identified in thisparticular tumor, and while this mutation has not been identified inhumans, research has indicated that this mutation, or a D351E mutation,causes many SERMs, including raloxifene, EM652, GW7604, keoxifene andtamoxifen, to have an estrogenic response. A mutation featured in theinvention, e.g., a mutation in the ligand binding domain in the vicinityof D351 may therefore indicate that a SERM should not be administeredfor treatment of the cancer, and an aromatase inhibitor should beadministered instead.

Other cancer-associated mutations identified in the ligand bindingdomain are believed to arise in response to treatment of a tumor with anestrogen mimetic, called a SERM (“Selective Estrogen ReceptorMoldulator”), such as tamoxifen. For example, a D351Y or D351E mutationresults in a receptor that exhibits an estrogenic response to therapywith SERMs including raloxifene (Evista®), EM652, GW7604, keoxifene,toremifene (Fareston®) and tamoxifen (Nolvadex®). Certain mutations inthe ligand binding domain have been found to result in resistance of thetumor to estrogen mimetic therapy (e.g., tamoxifen resistance). Thus,the identification of the 6 nucleotide deletion described herein, or anoverlapping mutation or a new mutation in the ligand binding domain ofESR1 can indicate the need to change therapies for a patient currentlyon a SERM therapy, or to alter the standard of care for the patient.

The identification of an ESR1 mutation as described herein may suggestthat a SERM should not be administered to a subject or that a subjectreceiving, or continuing to receive treatment with the SERM should bemonitored more frequently for worsening of cancer symptoms. In someembodiments, such as when the subject is a post-menopausal femalepatient, an anti-cancer agent that is not a SERM, e.g., an aromataseinhibitor, should be administered to the subject. When a subject is apre-menopausal female patient, identification of an ESR1 mutation asdescribed herein may suggest that the patient should receive analternative estrogen receptor blocking agent or an oophorectomy.

In one embodiment, a subject, e.g., a cancer patient, is alreadyreceiving therapy with a SERM, e.g., tamoxifen, and the identificationof a mutation in ESR1 may indicate that the patent should stop receivingtreatment with a SERM, or should receive a lower dose of SERM, or thatthe dose should be tapered (lowered over time).

A patient with a six nucleotide deletion as described herein, or anon-frameshift mutation that similarly deletes one or more of Leu, Ala,or Asp, at amino acid positions 349, 350 or 351 of SEQ ID NO:4,respectively, can be determined to not be a candidate to receive a SERMfor treatment of a cancer, or can be determined to be a candidate fortreatment with a lower dose of a SERM. A subject receiving or continuingto receive treatment with the SERM can optionally be monitored morefrequently for worsening of cancer symptoms. A post-menopausal patientwith a six nucleotide deletion as described herein, or a non-frameshiftmutation that similarly deletes one or more of Leu, Ala, or Asp, atamino acid positions of 349, 350 or 351 of SEQ ID NO:4, can bedetermined to be a candidate for treatment with an aromatase inhibitor.

Aromatase is the enzyme that synthesizes estrogen, and thus aromataseinhibitors are used to inhibit synthesis of estrogen in cancers thatrequire estrogen for growth. Exemplary aromatase inhibitors includenon-selective inhibitors, such as aminoglutethimide and testolactone(Teslac®); selective inhibitors, such as anastrozole (Arimidex®),letrozole (Femara®), exemestane (Aromasin®), vorozole (Rivizor),formestane (Lentaron®), and fadrozole (Afema); and the inhibitors4-hydroxyandrostenedione, 1,4,6-androstatrien-3,17-dione (ATD),4-Androstene-3,6,17-trione (“6-OXO”). Aromatase inhibitors includeirreversible steroidal inhibitors, such as exemestane and non-steroidalinhibitors, such as anastrozole.

In another embodiment, the identification of the 6 nucleotide deletiondescribed herein, or a similar mutation, can indicate that the patientshould stop administration with the current SERM therapy, and should beadministered the antiestrogen fulvestrant (Faslodex®), which has notbeen associated with SERM-resistant mutations in the region of aminoacid D351 of ESR1 (Herynk and Fuqua, Endocrine Reviews 25:869-898,2004).

In another embodiment, the identification of the 6 nucleotide deletiondescribed herein, or a similar mutation, can indicate that the patientshould stop administration with the current SERM therapy, and should beadministered the antiestrogen fulvestrant (Faslodex®) in combinationwith an aromatase therapy.

A pre-menopausal patient with a six nucleotide deletion as describedherein, or a non-frameshift mutation that similarly deletes one or moreof Leu, Ala, or Asp, at amino acid positions of 349, 350 or 351 of SEQID NO:4, can be determined to be a candidate for treatment with anoophorectomy (removal of the ovaries).

A novel mutation described herein is a 6 nucleotide deletion in theligand-binding domain of the ESR1 gene. The six nucleotide deletion isthe deletion of TGGCAG at nucleotides 1046-1051 of SEQ ID NO:1, whichresults in the deletion of amino acids LAD at position 349-351 of SEQ IDNO:1, and the insertion of histidine at position 349 of SEQ ID NO:2.

ESR1 is also known as estrogen receptor 1, estrogen receptor, estrogenreceptor a, estradiol receptor, Era, ER, ERα, ESR, ESRA, NR3A1, Nuclearreceptor subfamily 3 group A member 1, RP1-130E4_1, and DKFZp686N23123.The mRNA sequence is provided at NM_000125.3.

ESR1 is a nuclear hormone receptor that binds estrogen. The mainfunction of the estrogen receptor is as a DNA binding transcriptionfactor that regulates gene expression. A schematic diagram of the ESR1polypeptide is provided in FIG. 1. ESR1 contains two activation functiondomains (AF-1 and AF-2), a DNA binding domain, a hinge domain and aligand binding domain. AF-1 domain is phosphorylated at Ser116 andSer167, the DNA-binding domain is phosphorylated at Ser236, and the AF-2domain is phosphorylated at Tyr537. The AF-1 domain is aligand-independent transactivation domain, but the activation from AF-1is weak and more selective compared to the activation provided by thehormone-inducible transactivating function of AF-2. The AF-2 domaincontains the estrogen binding domain, as well as binding sites forcoactivator and corepressor proteins (Benecke et al., EMBO Reports1:151-157, 2000). Tamoxifen inhibits ESR1 activity by binding to theAF-2 domain (Id.). The DNA binding domain binds estrogen responseelements in DNA. At least six different ESR1 mRNA isoforms are generatedby alternative splicing and differ in their 5′ untranslated regions as aconsequence of alternative splicing of several upstream exons (1B-1F) toa common site 5′ to the translation initiation codon and thereforeresult in the generation of a common ER-α protein that is 66 kDa in size(Flouriot et al., EMBO J. 19:4688-4700, 1998). A seventh isoform(hERα46) lacks the N-terminal 173 amino acids of the full-length hERα66isoform, and inhibits hERα66 activity in a cell context where thetransactivating function of AF-1 predominates over AF-2.

In one embodiment, a mutation in the ligand binding domain includes anin-frame deletion that eliminates the aspartic acid at position 351 ofthe wildtype ESR1 protein. The in-frame deletion can delete three, six,nine, or twelve nucleotides or more, that results in the deletion ofone, two, three, four or five or more amino acids including the asparticacid at position 351 of SEQ ID NO:2. The nucleotide deletion can includenucleotides between and including 1030-1068 of SEQ ID NO:1, e.g.,between and including 1033-1065 of SEQ ID NO:1, e.g., between andincluding nucleotides 1036-1062 of SEQ ID NO:1. The deletion can resultin the deletion of amino acids, and optionally, the insertion of newamino acids in the mutant ESR1 protein. For example, amino acids atpositions from 344-356 of SEQ ID NO:2 can be deleted, e.g., frompositions 346-354 of SEQ ID NO:2, e.g., from 349-351 of SEQ ID NO:2. Thedeletion can further result in an amino acid insertion or substitution,e.g., at a position between 344-356 of SEQ ID NO:2, e.g., from positions346-354 of SEQ ID NO:2, e.g., from 349-351 of SEQ ID NO:2. In oneembodiment, a histidine is inserted at position 349.

Estrogen and estrogen receptor a have been implicated in breast cancer,ovarian cancer, endometrial cancer, prostate cancer and colon cancer.

Accordingly, the invention provides, at least in part, isolated ESR1nucleic acid molecules containing a mutation in the ligand bindingdomain, e.g., a 6 nucleotide deleting in the ligand binding domain asdescribed herein, nucleic acid constructs, host cells containing thenucleic acid molecules; purified mutant ESR1 polypeptides comprising amutation in the ligand binding domain, e.g., a deleting of the LAD atpositions 349-351 of SEQ ID NO:2 and the insertion of histidine atposition 349, and binding agents, e.g., antibodies and small moleculecompounds that specifically bind the mutant protein. The invention alsoprovides detection reagents (e.g., probes, primers, antibodies, kits);screening assays for identifying novel ESR1 inhibitors; as well asmethods, assays and kits for evaluating, identifying, assessing and/ortreating a subject having a cancer, e.g., a cancer having an ESR1mutation disclosed herein. The compositions and methods identifiedherein can be used, for example, to identify new ESR1 inhibitors; totreat or prevent a cancer; as well as in methods or assays forevaluating a cancer (e.g., evaluating one or more of: cancerprogression, cancer treatment response or resistance to cancertreatment; selecting a treatment option, stratifying a patientpopulation, and/or more effectively monitoring, treating or preventing acancer).

Other novel mutations are described in Table 1 and 2. The inventiontherefore also provides, at least in part, isolated nucleic acidmolecules containing a mutation in Table 1 or Table 1, nucleic acidconstructs, and host cells containing the nucleic acid molecules;purified mutant polypeptides comprising a mutation described in Table 1or Table 2, and binding agents, e.g., antibodies and small moleculecompounds that specifically bind the mutant proteins. The invention alsoprovides detection reagents (e.g., probes, primers, antibodies, kits);screening assays for identifying novel inhibitors; as well as methods,assays and kits for evaluating, identifying, assessing and/or treating asubject having a cancer, e.g., a cancer having a mutation disclosedherein.

Certain terms are first defined. Additional terms are defined throughoutthe specification.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity, or a value, e.g., a numericalvalue, by “directly acquiring” or “indirectly acquiring” the physicalentity or value. “Directly acquiring” means performing a process (e.g.,performing a synthetic or analytical method) to obtain the physicalentity or value. “Indirectly acquiring” refers to receiving the physicalentity or value from another party or source (e.g., a third partylaboratory that directly acquired the physical entity or value).Directly acquiring a physical entity includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial. Exemplary changes include making a physical entity from two ormore starting materials, shearing or fragmenting a substance, separatingor purifying a substance, combining two or more separate entities into amixture, performing a chemical reaction that includes breaking orforming a covalent or non-covalent bond. Directly acquiring a valueincludes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

“Acquiring a sequence” as the term is used herein, refers to obtainingpossession of a nucleotide sequence or amino acid sequence, by “directlyacquiring” or “indirectly acquiring” the sequence. “Directly acquiring asequence” means performing a process (e.g., performing a synthetic oranalytical method) to obtain the sequence, such as performing asequencing method (e.g., a Next Generation Sequencing (NGS) method).“Indirectly acquiring a sequence” refers to receiving information orknowledge of, or receiving, the sequence from another party or source(e.g., a third party laboratory that directly acquired the sequence).The sequence acquired need not be a full sequence, e.g., sequencing ofat least one nucleotide, or obtaining information or knowledge, thatidentifies an ESR1 mutation disclosed herein as being present in asubject constitutes acquiring a sequence.

Directly acquiring a sequence includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial, such as a tissue sample, e.g., a biopsy, or an isolatednucleic acid (e.g., DNA or RNA) sample. Exemplary changes include makinga physical entity from two or more starting materials, shearing orfragmenting a substance, such as a genomic DNA fragment; separating orpurifying a substance (e.g., isolating a nucleic acid sample from atissue); combining two or more separate entities into a mixture,performing a chemical reaction that includes breaking or forming acovalent or non-covalent bond. Directly acquiring a value includesperforming a process that includes a physical change in a sample oranother substance as described above.

“Acquiring a sample” as the term is used herein, refers to obtainingpossession of a sample, e.g., a tissue sample or nucleic acid sample, by“directly acquiring” or “indirectly acquiring” the sample. “Directlyacquiring a sample” means performing a process (e.g., performing aphysical method such as a surgery or extraction) to obtain the sample.“Indirectly acquiring a sample” refers to receiving the sample fromanother party or source (e.g., a third party laboratory that directlyacquired the sample). Directly acquiring a sample includes performing aprocess that includes a physical change in a physical substance, e.g., astarting material, such as a tissue, e.g., a tissue in a human patientor a tissue that has was previously isolated from a patient. Exemplarychanges include making a physical entity from a starting material,dissecting or scraping a tissue; separating or purifying a substance(e.g., a sample tissue or a nucleic acid sample); combining two or moreseparate entities into a mixture; performing a chemical reaction thatincludes breaking or forming a covalent or non-covalent bond. Directlyacquiring a sample includes performing a process that includes aphysical change in a sample or another substance, e.g., as describedabove.

“Binding entity” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of specifically bindingto an analyte. The binding entity can be an affinity tag on a nucleicacid sequence. In certain embodiments, the binding entity allows forseparation of the nucleic acid from a mixture, such as an avidinmolecule, or an antibody that binds to the hapten or an antigen-bindingfragment thereof. Exemplary binding entities include, but are notlimited to, a biotin molecule, a hapten, an antibody, an antibodybinding fragment, a peptide, and a protein.

“Complementary” refers to sequence complementarity between regions oftwo nucleic acid strands or between two regions of the same nucleic acidstrand. It is known that an adenine residue of a first nucleic acidregion is capable of forming specific hydrogen bonds (“base pairing”)with a residue of a second nucleic acid region which is antiparallel tothe first region if the residue is thymine or uracil. Similarly, it isknown that a cytosine residue of a first nucleic acid strand is capableof base pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.In certain embodiments, the first region comprises a first portion andthe second region comprises a second portion, whereby, when the firstand second portions are arranged in an antiparallel fashion, at leastabout 50%, at least about 75%, at least about 90%, or at least about 95%of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. In otherembodiments, all nucleotide residues of the first portion are capable ofbase pairing with nucleotide residues in the second portion.

The term “cancer” or “tumor” is used interchangeably herein. These termsrefer to the presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in theform of a tumor, but such cells can exist alone within an animal, or canbe a non-tumorigenic cancer cell, such as a leukemia cell. These termsinclude a solid tumor, a soft tissue tumor, or a metastatic lesion. Asused herein, the term “cancer” includes premalignant, as well asmalignant cancers.

Cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cancer isalso “inhibited” if recurrence or metastasis of the cancer is reduced,slowed, delayed, or prevented.

“Chemotherapeutic agent” means a chemical substance, such as a cytotoxicor cytostatic agent, that is used to treat a condition, particularlycancer.

As used herein, “cancer therapy” and “cancer treatment” are synonymousterms.

As used herein, “chemotherapy” and “chemotherapeutic” and“chemotherapeutic agent” are synonymous terms.

As used herein, “estrogen receptor (ER) positive (+)” refers to a samplethat contains an estrogen receptor, e.g., an ER detected by protein ornucleic acid levels. In one embodiment, a score of Estrogen Receptorpositive (ER+) means that estrogen is causing a detectable response in atumor, e.g., estrogen is causing a tumor to grow. In other embodiments,a score of Estrogen Receptor negative (ER−), means that estrogen is notcausing a tumor to grow. ER+ status can reflect an increased level oractivity of an ER relative to a reference sample (e.g., an ER− sample).

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence similarity between two polynucleotide sequences orbetween two polypeptide sequences, with identity being a more strictcomparison. The phrases “percent identity or homology” and “% identityor homology” refer to the percentage of sequence similarity found in acomparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value there between.Identity or similarity can be determined by comparing a position in eachsequence that can be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical or matching nucleotides at positions sharedby the polynucleotide sequences. A degree of identity of polypeptidesequences is a function of the number of identical amino acids atpositions shared by the polypeptide sequences. A degree of homology orsimilarity of polypeptide sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences. The term“substantially identical,” as used herein, refers to an identity orhomology of at least 75%, at least 80%, at least 85%, at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

“Likely to” or “increased likelihood,” as used herein, refers to anincreased probability that an item, object, thing or person will occur.Thus, in one example, a subject that is likely to respond to treatmentwith a SERM or an aromatase inhibitor, alone or in combination, has anincreased probability of responding to treatment with the SERM or thearomatase inhibitor alone or in combination, relative to a referencesubject or group of subjects.

“Unlikely to” refers to a decreased probability that an event, item,object, thing or person will occur with respect to a reference. Thus, asubject that is unlikely to respond to treatment with a SERM, alone orin combination, has a decreased probability of responding to treatmentwith a SERM, alone or in combination, relative to a reference subject orgroup of subjects.

“Sequencing” a nucleic acid molecule requires determining the identityof at least 1 nucleotide in the molecule. In embodiments, the identityof less than all of the nucleotides in a molecule are determined. Inother embodiments, the identity of a majority or all of the nucleotidesin the molecule is determined.

“Next-generation sequencing or NGS or NG sequencing” as used herein,refers to any sequencing method that determines the nucleotide sequenceof either individual nucleic acid molecules (e.g., in single moleculesequencing) or clonally expanded proxies for individual nucleic acidmolecules in a highly parallel fashion (e.g., greater than 10⁵ moleculesare sequenced simultaneously). In one embodiment, the relative abundanceof the nucleic acid species in the library can be estimated by countingthe relative number of occurrences of their cognate sequences in thedata generated by the sequencing experiment. Next generation sequencingmethods are known in the art, and are described, e.g., in Metzker, M.(2010) Nature Biotechnology Reviews 11:31-46, incorporated herein byreference. Next generation sequencing can detect a variant present inless than 5% of the nucleic acids in a sample.

“Sample,” “tissue sample,” “patient sample,” “patient cell or tissuesample” or “specimen” each refers to a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample can be solid tissue as from a fresh, frozen and/or preservedorgan, tissue sample, biopsy, or aspirate; blood or any bloodconstituents; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid or interstitial fluid; or cells from any time ingestation or development of the subject. The tissue sample can containcompounds that are not naturally intermixed with the tissue in naturesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics or the like. In one embodiment, the sample is preserved as afrozen sample or as formaldehyde- or paraformaldehyde-fixedparaffin-embedded (FFPE) tissue preparation. For example, the sample canbe embedded in a matrix, e.g., an FFPE block or a frozen sample.

A “tumor nucleic acid sample” as used herein, refers to nucleic acidmolecules from a tumor or cancer sample. Typically, it is DNA, e.g.,genomic DNA, or cDNA derived from RNA, from a tumor or cancer sample. Incertain embodiments, the tumor nucleic acid sample is purified orisolated (e.g., it is removed from its natural state).

A “control” or “reference” “nucleic acid sample” as used herein, refersto nucleic acid molecules from a control or reference sample. Typically,it is DNA, e.g., genomic DNA, or cDNA derived from RNA, not containingthe alteration or variation in the gene or gene product, e.g., notcontaining a mutation in the ESR1 gene. In certain embodiments, thereference or control nucleic acid sample is a wildtype or a non-mutatedsequence. In certain embodiments, the reference nucleic acid sample ispurified or isolated (e.g., it is removed from its natural state). Inother embodiments, the reference nucleic acid sample is from a non-tumorsample, e.g., a blood control, a normal adjacent tumor (NAT), or anyother non-cancerous sample from the same or a different subject.

“Adjacent to the interrogation position,” as used herein, means that asite sufficiently close such that a detection reagent complementary withthe site can be used to distinguish between a mutation, e.g., a mutationdescribed herein, and a reference sequence, e.g., a non-mutant orwild-type sequence, in a target nucleic acid. Directly adjacent, as usedherein, is where 2 nucleotides have no intervening nucleotides betweenthem.

“Associated mutation,” as used herein, refers to a mutation within apreselected distance, in terms of nucleotide or primary amino acidsequence, from a definitional mutation, e.g., a mutant as describedherein, e.g., a mutation in Table 3. In embodiments, the associatedmutation is within n, wherein n is 2, 5, 10, 20, 30, 50, 100, or 200nucleotides from the definitional mutation (n does not include thenucleotides defining the associated and definitional mutations). Inembodiments the associated mutation is a missense mutation or anin-frame deletion or insertion.

“Interrogation position,” as used herein, comprises at least onenucleotide (or, in the case of polypeptides, an amino acid residue)which corresponds to a nucleotide (or amino acid residue) that ismutated in a mutation of interest, e.g., a mutation being identified, orin a nucleic acid (or protein) being analyzed, e.g., sequenced, orrecovered. By way of example the interrogation position in the T311Mmutation described herein includes nucleotide position 932 and theinterrogation position in the deletion at 1046-1051 includes one or moreof nucleotide positions 1046-1051.

A “reference sequence,” as used herein, e.g., as a comparator for amutant sequence, is a sequence which has a different nucleotide or aminoacid at an interrogation position than does the mutant(s) beinganalyzed. In an embodiment the reference sequence is wild-type for atleast the interrogation position.

Headings, e.g., (a), (b), (i) etc, are presented merely for ease ofreading the specification and claims. The use of headings in thespecification or claims does not require the steps or elements beperformed in alphabetical or numerical order or the order in which theyare presented.

Various aspects of the invention are described in further detail below.Additional definitions are set out throughout the specification.

Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat include an ESR1 mutation, including nucleic acids which encode anESR1 polypeptide that contains a mutation in the ligand binding domain,or a portion of such an ESR1 polypeptide. The nucleic acid moleculesinclude those nucleic acid molecules which reside in genomic regionsidentified herein. As used herein, the term “nucleic acid molecule”includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded; in certain embodiments the nucleic acid molecule isdouble-stranded DNA.

Isolated nucleic acid molecules also include nucleic acid moleculessufficient for use as hybridization probes or primers to identifynucleic acid molecules that contain a mutation in an ESR1 gene, e.g., inthe ligand binding domain of an ESR1 gene, e.g., nucleic acid moleculessuitable for use as PCR primers for the amplification or mutation ofnucleic acid molecules.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. In certain embodiments, an “isolated” nucleicacid molecule is free of sequences (such as protein-encoding sequences)which naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kB, less than about 4 kB, less than about 3 kB, less than about2 kB, less than about 1 kB, less than about 0.5 kB or less than about0.1 kB of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

The language “substantially free of other cellular material or culturemedium” includes preparations of nucleic acid molecule in which themolecule is separated from cellular components of the cells from whichit is isolated or recombinantly produced. Thus, nucleic acid moleculethat is substantially free of cellular material includes preparations ofnucleic acid molecule having less than about 30%, less than about 20%,less than about 10%, or less than about 5% (by dry weight) of othercellular material or culture medium.

A nucleic acid molecule containing a mutation in the ESR1 gene can beisolated using standard molecular biology techniques and the sequenceinformation in the database records described herein. Using all or aportion of such nucleic acid sequences, mutant ESR1 nucleic acidmolecules can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook et al., ed., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule containing a mutation in the ESR1 gene, e.g., inthe ligand binding domain of ESR1 can be amplified using cDNA, mRNA, orgenomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acidmolecules so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In one embodiment, a nucleic acid molecule containing an ESR1 mutationcomprises a nucleic acid molecule which has a nucleotide sequencecomplementary to the nucleotide sequence of a mutant ESR1 nucleic acidmolecule or to the nucleotide sequence of a nucleic acid encoding amutant ESR1 protein. A nucleic acid molecule which is complementary to agiven nucleotide sequence is one which is sufficiently complementary tothe given nucleotide sequence that it can hybridize to the givennucleotide sequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule containing a mutation in the ESR1gene, e.g., in the ligand binding domain of the ESR1 gene, can compriseonly a portion of a nucleic acid sequence, wherein the full lengthnucleic acid sequence encodes a mutant ESR1 polypeptide. Such nucleicacid molecules can be used, for example, as a probe or primer. Theprobe/primer typically is used as one or more substantially purifiedoligonucleotides. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 7, at least about 15, at least about 25, at least about 50,at least about 75, at least about 100, at least about 125, at leastabout 150, at least about 175, at least about 200, at least about 250,at least about 300, at least about 350, at least about 400, at leastabout 500, at least about 600, at least about 700, at least about 800,at least about 900, at least about 1 kb, at least about 2 kb, at leastabout 3 kb, at least about 4 kb, at least about 5 kb, at least about 6kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, atleast about 10 kb, at least about 15 kb, at least about 20 kb, at leastabout 25 kb, at least about 30 kb, at least about 35 kb, at least about40 kb, at least about 45 kb, at least about 50 kb, at least about 60 kb,at least about 70 kb, at least about 80 kb, at least about 90 kb, atleast about 100 kb, at least about 200 kb, at least about 300 kb, atleast about 400 kb, at least about 500 kb, at least about 600 kb, atleast about 700 kb, at least about 800 kb, at least about 900 kb, atleast about 1 mb, at least about 2 mb, at least about 3 mb, at leastabout 4 mb, at least about 5 mb, at least about 6 mb, at least about 7mb, at least about 8 mb, at least about 9 mb, at least about 10 mb ormore consecutive nucleotides of a mutant ESR1 nucleic acid. The mutantnucleic acid can include a fusion junction created from the deletion ofpart of the ESR1 gene, e.g., part of the ligand binding domain of theESR1 gene.

The invention further encompasses nucleic acid molecules that aresubstantially identical to the gene mutations and/or gene productsdescribed herein, e.g., a mutant ESR1 gene having a nucleotide sequenceof SEQ ID NO:3 (or an amino acid sequence of SEQ ID NO: 4) such thatthey are at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5% orgreater. In other embodiments, the invention further encompasses nucleicacid molecules that are substantially homologous to the ESR1 mutant geneand/or gene products described herein, such that they differ by only orat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90, at least 100, atleast 200, at least 300, at least 400, at least 500, at least 600nucleotides or any range in between.

In another embodiment, an isolated mutant ESR1 nucleic acid molecule isat least 7, at least 15, at least 20, at least 25, at least 30, at least35, at least 40, at least 45, at least 50, at least 55, at least 60, atleast 65, at least 70, at least 75, at least 80, at least 85, at least90, at least 95, at least 100, at least 125, at least 150, at least 175,at least 200, at least 250, at least 300, at least 350, at least 400, atleast 450, at least 550, at least 650, at least 700, at least 800, atleast 900, at least 1000, at least 1200, at least 1400, at least 1600,at least 1800, at least 2000, at least 2200, at least 2400, at least2600, at least 2800, at least 3000, or more nucleotides in length andhybridizes under stringent conditions to a mutant ESR1 nucleic acidmolecule or to a nucleic acid molecule encoding a protein correspondingto a marker of the invention.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, or at least 85% identical to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in sections6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989). Another, non-limiting example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C.

The invention also includes molecular beacon nucleic acid moleculeshaving at least one region which is complementary to a mutant ESR1nucleic acid molecule, such that the molecular beacon is useful forquantitating the presence of the nucleic acid molecule of the inventionin a sample. A “molecular beacon” nucleic acid is a nucleic acidmolecule comprising a pair of complementary regions and having afluorophore and a fluorescent quencher associated therewith. Thefluorophore and quencher are associated with different portions of thenucleic acid in such an orientation that when the complementary regionsare annealed with one another, fluorescence of the fluorophore isquenched by the quencher. When the complementary regions of the nucleicacid molecules are not annealed with one another, fluorescence of thefluorophore is quenched to a lesser degree. Molecular beacon nucleicacid molecules are described, for example, in U.S. Pat. No. 5,876,930.

Probes

The invention also provides isolated ESR1 mutant nucleic acid moleculesuseful as probes.

Probes based on the sequence of a mutant ESR1 nucleic acid molecule canbe used to detect transcripts or genomic sequences corresponding to oneor more markers of the invention. The probe comprises a label groupattached thereto, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as part of atest kit for identifying cells or tissues which express the mutant ESR1protein, such as by measuring levels of a nucleic acid molecule encodingthe protein in a sample of cells from a subject, e.g., detecting mRNAlevels or determining whether a gene encoding the protein has beenmutated or deleted.

Probes featured in the invention include those that will specificallyhybridize to a gene sequence described in the Example, e.g., an ESR1gene having a six nucleotide deletion in the ligand binding domain.Typically these probes are 12 to 20, e.g., 17 to 20 nucleotides inlength (longer for large insertions) and have the nucleotide sequencecorresponding to the region of the mutations at their respectivenucleotide locations on the gene sequence. Such molecules can be labeledaccording to any technique known in the art, such as with radiolabels,fluorescent labels, enzymatic labels, sequence tags, biotin, otherligands, etc. As used herein, a probe that “specifically hybridizes” toan ESR1 mutant gene sequence will hybridize under high stringencyconditions.

A probe will typically contain one or more of the specific mutationsdescribed herein. Typically, a nucleic acid probe will encompass onlyone mutation. Such molecules may be labeled and can be used asallele-specific probes to detect the mutation of interest.

In one aspect, the invention features a probe or probe set thatspecifically hybridizes to a nucleic acid comprising a deletion in theligand binding domain of ESR1.

Isolated pairs of allele specific oligonucleotide probes are alsoprovided, where the first probe of the pair specifically hybridizes tothe mutant allele, and the second probe of the pair specificallyhybridizes to the wildtype allele. For example, in one exemplary probepair, one probe will recognize the junction created by the deletion inthe ligand binding domain of the ESR1 gene, and the other probe willrecognize a sequence downstream or upstream of the deletion. Theseallele-specific probes are useful in detecting an ESR1 somatic mutationin a tumor sample, e.g., a breast tumor sample.

Primers

The invention also provides isolated nucleic acid molecules useful asprimers.

The term “primer” as used herein refers to a sequence comprising two ormore deoxyribonucleotides or ribonucleotides, e.g., more than three, andmore than eight, or at least 20 nucleotides of a gene described in theExample, where the sequence corresponds to a sequence flanking one ofthe mutations or a wildtype sequence of a gene identified in theExample, e.g., an ESR1 gene. Primers may be used to initiate DNAsynthesis via the PCR (polymerase chain reaction) or a sequencingmethod. Primers featured in the invention include the sequences recitedand complementary sequences which would anneal to the opposite DNAstrand of the sample target. Since both strands of DNA are complementaryand mirror images of each other, the same segment of DNA will beamplified.

Primers can be used to sequence a nucleic acid, e.g., an isolatednucleic acid described herein, such as by an NGS method, or to amplify agene described in the Example, such as by PCR. The primers canspecifically hybridize, for example, to the ends of the exons or to theintrons flanking the exons. The amplified segment can then be furtheranalyzed for the presence of the mutation such as by a sequencingmethod, or by a size separation technique such as by electrophoresis ona gel. The primers are useful in directing amplification of a targetpolynucleotide prior to sequencing. In another aspect, the inventionfeatures a pair of oligonucleotide primers that amplify a region thatcontains or is adjacent to a fusion junction identified in FIG. 4B. Suchprimers are useful in directing amplification of a target region thatincludes a fusion junction identified in the FIG. 4B, e.g., prior tosequencing. The primer typically contains 12 to 20, or 17 to 20, or morenucleotides, although a primer may contain fewer nucleotides.

A primer is typically single stranded, e.g., for use in sequencing oramplification methods, but may be double stranded. If double stranded,the primer may first be treated to separate its strands before beingused to prepare extension products. A primer must be sufficiently longto prime the synthesis of extension products in the presence of theinducing agent for polymerization. The exact length of primer willdepend on many factors, including applications (e.g., amplificationmethod), temperature, buffer, and nucleotide composition. A primertypically contains 12-20 or more nucleotides, although a primer maycontain fewer nucleotides.

Primers are typically designed to be “substantially” complementary toeach strand of a genomic locus to be amplified. Thus, the primers mustbe sufficiently complementary to specifically hybridize with theirrespective strands under conditions which allow the agent forpolymerization to perform. In other words, the primers should havesufficient complementarity with the 5′ and 3′ sequences flanking themutation to hybridize therewith and permit amplification of the genomiclocus.

The term “substantially complementary to” or “substantially thesequence” refers to sequences that hybridize to the sequences providedunder stringent conditions and/or sequences having sufficient homologywith a sequence comprising a fusion junction identified in FIG. 4B, orthe wildtype counterpart sequence, such that the allele specificoligonucleotides hybridize to the sequence. In one embodiment, asequence is substantially complementary to a fusion junction created bya deletion event, e.g., to a fusion junction in SEQ ID NO:3.“Substantially the same” as it refers to oligonucleotide sequences alsorefers to the functional ability to hybridize or anneal with sufficientspecificity to distinguish between the presence or absence of themutation. This is measurable by the temperature of melting beingsufficiently different to permit easy identification of whether theoligonucleotide is binding to the normal or mutant gene sequenceidentified in the Example.

In one aspect, the invention features a primer or primer set foramplifying a nucleic acid comprising a deletion resulting in a ESR1mutation.

Isolated pairs of allele specific oligonucleotide primer are alsoprovided, where the first primer of the pair specifically hybridizes tothe mutant allele, and the second primer of the pair specificallyhybridizes to a sequence upstream or downstream of a mutation, or afusion junction resulting from, e.g., an inversion, duplication,deletion, insertion or translocation. For example, in one exemplaryprimer pair, one probe will recognize an ESR1 mutation, such as byhybridizing to a sequence at the fusion junction resulting from thenucleotide(s) deletion, and the other primer will recognize a sequenceupstream or downstream of the fusion junction. These allele-specificprimers are useful for amplifying a mutant ESR1 sequence from a tumorsample, e.g., a breast tumor sample.

Primers can be prepared using any suitable method, such as conventionalphosphotriester and phosphodiester methods or automated embodimentsthereof. In one such automated embodiment, diethylphosphoramidites areused as starting materials and may be synthesized as described byBeaucage, et al., Tetrahedron Letters, 22:1859-1862, (1981). One methodfor synthesizing oligonucleotides on a modified solid support isdescribed in U.S. Pat. No. 4,458,066.

An oligonucleotide probe or primer that hybridizes to a mutant orwildtype allele is said to be the complement of the allele. As usedherein, a probe exhibits “complete complementarity” when everynucleotide of the probe is complementary to the corresponding nucleotideof the allele. Two polynucleotides are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, thepolynucleotides are said to be “complementary” if they can hybridize toone another with sufficient stability to permit them to remain annealedto one another under conventional “high-stringency” conditions.Conventional stringency conditions are known to those skilled in the artand can be found, for example in Molecular Cloning: A Laboratory Manual,3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N.Irwin, Cold Spring Harbor Laboratory Press, 2000.

Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of aprobe to hybridize to an allele. Thus, in order for a polynucleotide toserve as a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed. Appropriatestringency conditions which promote DNA hybridization are, for example,6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by awash of 2.0×SSC at 50° C. Such conditions are known to those skilled inthe art and can be found, for example in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989). Salt concentration andtemperature in the wash step can be adjusted to alter hybridizationstringency. For example, conditions may vary from low stringency ofabout 2.0×SSC at 40° C. to moderately stringent conditions of about2.0×SSC at 50° C. to high stringency conditions of about 0.2×SSC at 50°C.

ESR1 Mutant Proteins and Antibodies

One aspect of the invention pertains to purified ESR1 mutantpolypeptides, and biologically active portions thereof. In oneembodiment, the native ESR1 mutant polypeptide can be isolated fromcells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. In another embodiment, an ESR1mutant polypeptide is produced by recombinant DNA techniques.Alternative to recombinant expression, a mutant ESR1 polypeptide can besynthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, less than about 20%, less than about 10%, orless than about 5% (by dry weight) of heterologous protein (alsoreferred to herein as a “contaminating protein”). When the protein orbiologically active portion thereof is recombinantly produced, it can besubstantially free of culture medium, i.e., culture medium representsless than about 20%, less than about 10%, or less than about 5% of thevolume of the protein preparation. When the protein is produced bychemical synthesis, it can be substantially free of chemical precursorsor other chemicals, i.e., it is separated from chemical precursors orother chemicals which are involved in the synthesis of the protein.Accordingly such preparations of the protein have less than about 30%,less than about 20%, less than about 10%, less than about 5% (by dryweight) of chemical precursors or compounds other than the polypeptideof interest.

Biologically active portions of an ESR1 mutant polypeptide includepolypeptides comprising amino acid sequences sufficiently identical toor derived from the amino acid sequence of the mutant ESR1 protein,which include fewer amino acids than the full length protein, andexhibit at least one activity of the corresponding full-length protein,e.g., a ligand-binding activity. A biologically active portion of aprotein of the invention can be a polypeptide which is, for example, 10,25, 50, 100 or more amino acids in length. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe functional activities of the native form of a polypeptide.

In certain embodiments, the ESR1 mutant polypeptide has an amino acidsequence of a protein encoded by a nucleic acid molecule disclosedherein. Other useful proteins are substantially identical (e.g., atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 86, at least 87, at least 88, at least 89, at least 90, atleast 91, at least 92, at least 93, at least 94, at least 95, at least96, at least 97, at least 98, at least 99, at least 99.5% or greater) toone of these sequences and retain the functional activity of the proteinof the corresponding full-length protein yet differ in amino acidsequence.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. Another, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Suchan algorithm is incorporated into the ALIGN program (version 2.0) whichis part of the GCG sequence alignment software package. When utilizingthe ALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. Yet another useful algorithm for identifying regions of localsequence similarity and alignment is the FASTA algorithm as described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. Whenusing the FASTA algorithm for comparing nucleotide or amino acidsequences, a PAM120 weight residue table can, for example, be used witha k-tuple value of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

An isolated ESR1 mutant polypeptide, or a fragment thereof, can be usedas an immunogen to generate antibodies using standard techniques forpolyclonal and monoclonal antibody preparation. The full-length ESR1mutant polypeptide can be used or, alternatively, the invention providesantigenic peptide fragments for use as immunogens. The antigenic peptideof a protein of the invention comprises at least 8 (or at least 10, atleast 15, at least 20, or at least 30 or more) amino acid residues ofthe amino acid sequence of one of the polypeptides of the invention, andencompasses an epitope of the protein such that an antibody raisedagainst the peptide forms a specific immune complex with a marker of theinvention to which the protein corresponds. Exemplary epitopesencompassed by the antigenic peptide are regions that are located on thesurface of the protein, e.g., hydrophilic regions. Hydrophobicitysequence analysis, hydrophilicity sequence analysis, or similar analysescan be used to identify hydrophilic regions.

An immunogen typically is used to prepare antibodies by immunizing asuitable (i.e., immunocompetent) subject such as a rabbit, goat, mouse,or other mammal or vertebrate. An appropriate immunogenic preparationcan contain, for example, recombinantly-expressed orchemically-synthesized polypeptide. The preparation can further includean adjuvant, such as Freund's complete or incomplete adjuvant, or asimilar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodiesdirected against a mutant ESR1 polypeptide. In one embodiment, theantibody molecule specifically binds to the junction created by thedeletion in the ligand binding domain, e.g., specifically binds to anepitope formed as a result of the nucleotide deletion. In someembodiments the antibody can distinguish wildtype ESR1 from mutant ESR1.

The terms “antibody” and “antibody molecule” as used interchangeablyherein refer to immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site which specifically binds an antigen, such as apolypeptide of the invention. A molecule which specifically binds to agiven polypeptide of the invention is a molecule which binds thepolypeptide, but does not substantially bind other molecules in asample, e.g., a biological sample, which naturally contains thepolypeptide. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition,” as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide of the invention as an immunogen.Antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (see Kozbor etal., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Coleet al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., 1985) or trioma techniques. The technology for producinghybridomas is well known (see generally Current Protocols in Immunology,Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindthe polypeptide of interest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the polypeptide of interest. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions canbe made using standard recombinant DNA techniques. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described in PCTPublication No. WO 87/02671; European Patent Application 184,187;European Patent Application 171,496; European Patent Application173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Application 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559; Morrison (1985)Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat.No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

Completely human antibodies can be produced using transgenic mice whichare incapable of expressing endogenous immunoglobulin heavy and lightchains genes, but which can express human heavy and light chain genes.For an overview of this technology for producing human antibodies, seeLonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

An antibody directed against a mutant ESR1 polypeptide (e.g., amonoclonal antibody) can be used to isolate the polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation.Moreover, such an antibody can be used to detect the marker (e.g., in acellular lysate or cell supernatant) in order to evaluate the level andpattern of expression of the marker. The antibodies can also be useddiagnostically to monitor protein levels in tissues or body fluids(e.g., in a tumor cell-containing body fluid) as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude, but are not limited to, various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes include, but arenot limited to, horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include, but are not limited to,streptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include, but are not limited to, umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes, but is not limited to, luminol; examplesof bioluminescent materials include, but are not limited to, luciferase,luciferin, and aequorin, and examples of suitable radioactive materialsinclude, but are not limited to, ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

Antigens and Vaccines

Embodiments of the invention include preparations, e.g., antigenicpreparations, of the entire mutant ESR1 or a fragment thereof, e.g., afragment capable of raising antibodies specific to the fusion junctioncreated by the deletion in the ligand binding domain of ESR1(collectively referred to herein as a mutant specific polypeptides orMSP). The preparation can include an adjuvant or other component.

An MSP can be used as an antigen or vaccine. For example, an MSP can beused as an antigen to immunize an animal, e.g., a rodent, e.g., a mouseor rat, rabbit, horse, goat, dog, or non-human primate, to obtainantibodies, e.g., mutant protein specific antibodies. In an embodiment amutant specific antibody molecule is an antibody molecule describedherein, e.g., a polyclonal. In other embodiments a mutant specificantibody molecule is monospecific, e.g., monoclonal, human, humanized,chimeric or other monospecific antibody molecule. The mutant proteinspecific antibody molecules can be used to treat a subject havingcancer, e.g., a cancer described herein.

Embodiments of the invention include vaccine preparations that comprisean MSP capable of stimulating an immune response in a subject, e.g., byraising, in the subject, antibodies specific to the mutant protein. Thevaccine preparation can include other components, e.g., an adjuvant. Thevaccine preparations can be used to treat a subject having cancer, e.g.,a cancer described herein.

In other embodiments, the preparations include the entire mutant ESR1gene, or a fragment comprising the mutation in ESR1. The mutation can bean amino acid substitution or insertion as well as a deletion.

Expression Vectors, Host Cells and Recombinant Cells

In another aspect, the invention includes vectors (e.g., expressionvectors), containing a nucleic acid encoding a mutant ESR1 polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a mutant ERS1 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Typically, therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorscan be introduced into host cells to thereby produce the mutant ESR1polypeptide, including proteins or polypeptides encoded by nucleic acidsas described herein, mutant forms thereof, and the like).

The term “recombinant host cell” (or simply “host cell” or “recombinantcell”), as used herein, is intended to refer to a cell into which arecombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell, but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The recombinant expression vectors can be designed for expression of amutant ESR1 polypeptide in prokaryotic or eukaryotic cells. For example,polypeptides of the invention can be expressed in E. coli, insect cells(e.g., using baculovirus expression vectors), yeast cells or mammaliancells. Suitable host cells are discussed further in Goeddel, (1990) GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either mutant or wildtype proteins. Mutantvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such mutantvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the added amino acids andthe recombinant protein to enable separation of the recombinant proteinfrom the fusion moiety subsequent to purification of the mutant protein.Such enzymes, and their cognate recognition sequences, include FactorXa, thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

Purified mutant ESR1 polypeptides can be used in activity assays (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for mutant ESR1 polypeptides.

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman, S., (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.119-128). Another strategy is to alter the nucleic acid sequence of thenucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The mutant ESR1 polypeptide expression vector can be a yeast expressionvector, a vector for expression in insect cells, e.g., a baculovirusexpression vector or a vector suitable for expression in mammaliancells.

When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40.

In another embodiment, the promoter is an inducible promoter, e.g., apromoter regulated by a steroid hormone, by a polypeptide hormone (e.g.,by means of a signal transduction pathway), or by a heterologouspolypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and“Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy9:983).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example, the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a mutant ESR1 nucleic acidmolecule within a recombinant expression vector or a mutant ESR1 nucleicacid molecule containing sequences which allow it to homologousrecombination into a specific site of the host cell's genome.

A host cell can be any prokaryotic or eukaryotic cell. For example, amutant ESR1 polypeptide can be expressed in bacterial cells (such as E.coli), insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells, e.g., COS-7 cells, CV-1 origin SV40cells; Gluzman (1981) Cell 23:175-182). Other suitable host cells areknown to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell can be used to produce (e.g., express) a mutant ESR1polypeptide. Accordingly, the invention further provides methods forproducing a mutant ESR1 polypeptide using the host cells. In oneembodiment, the method includes culturing the host cell of the invention(into which a recombinant expression vector encoding a mutant ESR1polypeptide has been introduced) in a suitable medium such that a mutantESR1 polypeptide is produced. In another embodiment, the method furtherincludes isolating a mutant ESR1 polypeptide from the medium or the hostcell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a mutant ESR1 transgene, or whichotherwise misexpress mutant ESR1. The cell preparation can consist ofhuman or non-human cells, e.g., rodent cells, e.g., mouse or rat cells,rabbit cells, or pig cells. In embodiments, the cell or cells include amutant ESR1 transgene, e.g., a heterologous form of the mutant ESR1,e.g., a gene derived from humans (in the case of a non-human cell). Themutant ESR1 transgene can be misexpressed, e.g., overexpressed orunderexpressed. In other embodiments, the cell or cells include a genethat mis-expresses an endogenous mutant ESR1, e.g., a gene theexpression of which is disrupted, e.g., a knockout. Such cells can serveas a model for studying disorders that are related to mutated ormis-expressed ESR1 alleles (e.g., cancers) or for use in drug screening,as described herein.

Therapeutic Methods

Alternatively, or in combination with the methods described herein, theinvention features a method of treating a cancer or tumor harboring amutant ESR1 gene as described herein. The methods include administeringan anti-cancer agent, e.g., an SERM or an aromatase, alone or incombination, e.g., in combination with other chemotherapeutic agents orprocedures, in an amount sufficient to reduce or inhibit the tumor cellgrowth, and/or treat or prevent the cancer(s), in the subject.

“Treat,” “treatment,” and other forms of this word refer to theadministration of a SERM or an aromatase, alone or in combination with asecond agent to impede growth of a cancer, to cause a cancer to shrinkby weight or volume, to extend the expected survival time of the subjectand or time to progression of the tumor or the like. In those subjects,treatment can include, but is not limited to, inhibiting tumor growth,reducing tumor mass, reducing size or number of metastatic lesions,inhibiting the development of new metastatic lesions, prolongedsurvival, prolonged progression-free survival, prolonged time toprogression, and/or enhanced quality of life.

As used herein, unless otherwise specified, the terms “prevent,”“preventing” and “prevention” contemplate an action that occurs before asubject begins to suffer from the re-growth of the cancer and/or whichinhibits or reduces the severity of the cancer.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of the cancer, or todelay or minimize one or more symptoms associated with the cancer. Atherapeutically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other therapeuticagents, which provides a therapeutic benefit in the treatment ormanagement of the cancer. The term “therapeutically effective amount”can encompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of the cancer, or enhances the therapeutic efficacyof another therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to preventre-growth of the cancer, or one or more symptoms associated with thecancer, or prevent its recurrence. A prophylactically effective amountof a compound means an amount of the compound, alone or in combinationwith other therapeutic agents, which provides a prophylactic benefit inthe prevention of the cancer. The term “prophylactically effectiveamount” can encompass an amount that improves overall prophylaxis orenhances the prophylactic efficacy of another prophylactic agent.

As used herein, the term “patient” or “subject” refers to an animal,typically a human (i.e., a male or female of any age group, e.g., apediatric patient (e.g, infant, child, adolescent) or adult patient(e.g., young adult, middle-aged adult or senior adult) or other mammal,such as a primate (e.g., cynomolgus monkey, rhesus monkey); commerciallyrelevant mammals such as cattle, pigs, horses, sheep, goats, cats,and/or dogs; and/or birds, including commercially relevant birds such aschickens, ducks, geese, and/or turkeys, that will be or has been theobject of treatment, observation, and/or experiment. When the term isused in conjunction with administration of a compound or drug, then thepatient has been the object of treatment, observation, and/oradministration of the compound or drug.

In certain embodiments, the cancer includes, but is not limited to, asolid tumor, a soft tissue tumor, and a metastatic lesion (e.g., acancer as described herein). In one embodiment, the cancer is chosenfrom a breast cancer, a prostate cancer, an endometrial cancer, anovarian cancer, and a colon cancer.

In other embodiments, the cancer is chosen from lung cancer, thyroidcancer, colorectal cancer, adenocarcinoma, melanoma, B cell cancer,bronchus cancer, cancer of the oral cavity or pharynx, cancer ofhematological tissues, cervical cancer, esophageal cancer,esophageal-gastric cancer, gastric cancer, kidney cancer, liver cancer,multiple myeloma, pancreatic cancer, salivary gland cancer, small bowelor appendix cancer, stomach cancer, testicular cancer, urinary bladdercancer, uterine cancer, inflammatory myofibroblastic tumors,gastrointestinal stromal tumor (GIST), and the like.

In one embodiment, the anti-cancer agent is an SERM (“Selective EstrogenReceptor Modulator”). For example, the SERM can be chosen fromraloxifene (Evista®), EM652, GW7604, keoxifene, toremifene (Fareston®),tamoxifen (Nolvadex®), lasofoxifene, levormeloxifene, bazedoxifene, orarzoxifene. In another embodiment the anti-cancer agent is the estrogenantagonist fulvestrant (ICI 182, 780; Faslodex®), which also acts topromote degradation of the estrogen rector.

In another embodiment the anti-cancer agent is an aromatase inhibitorchosen from aminoglutethimide, testolactone (Teslac®), anastrozole(Arimidex®), letrozole (Femara®), exemestane (Aromasin®), vorozole(Rivizor), formestane (Lentaron®), fadrozole (Afema);4-hydroxyandrostenedione, 1,4,6-androstatrien-3,17-dione (ATD), and4-Androstene-3,6,17-trione (“6-OXO”).

In other embodiments, the anti-cancer agent is an ESR1 antagonist thatinhibits the expression of nucleic acid encoding a mutant ESR1, such asa mutant ESR1 described herein. Examples of such antagonists includenucleic acid molecules, for example, antisense molecules, ribozymes,RNAi, triple helix molecules that hybridize to a nucleic acid encodingESR1, or a transcription regulatory region, and blocks or reduces mRNAexpression of mutant ESR1.

In other embodiments, the SERM, aromatase inhibitor or other estrogeninhibitor is administered in combination with a second therapeutic agentor a different therapeutic modality, e.g., anti-cancer agents, and/or incombination with surgical and/or radiation procedures. In otherembodiments, the SERM, aromatase inhibitor or other estrogen inhibitoris administered in combination with a second therapeutic agent or adifferent therapeutic modality, e.g., to treat a symptom of chemotherapysuch as for treatment of nausea or headache.

By “in combination with,” it is not intended to imply that the therapyor the therapeutic agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. The pharmaceutical compositions canbe administered concurrently with, prior to, or subsequent to, one ormore other additional therapies or therapeutic agents. In general, eachagent will be administered at a dose and/or on a time scheduledetermined for that agent. It will further be appreciated that theadditional therapeutic agent utilized in this combination can beadministered together in a single composition or administered separatelyin different compositions. The particular combination to employ in aregimen will take into account compatibility of the inventivepharmaceutical composition with the additional therapeutically activeagent and/or the desired therapeutic effect to be achieved.

For example, the second therapeutic agent can be a cytotoxic or acytostatic agent. Exemplary cytotoxic agents include antimicrotubuleagents, topoisomerase inhibitors, or taxanes, antimetabolites, mitoticinhibitors, alkylating agents, intercalating agents, agents capable ofinterfering with a signal transduction pathway, agents that promoteapoptosis and radiation. In yet other embodiments, the methods can beused in combination with immunodulatory agents, e.g., IL-1, 2, 4, 6, or12, or interferon α or γ, or immune cell growth factors such as GM-CSF.

Anti-cancer agents, e.g., estrogen inhibitors such as SERMs andaromatase inhibitors, used in the therapeutic methods featured in theinvention can be evaluated using the screening assays described herein.In one embodiment, the anti-cancer agents are evaluated in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the anti-cancer agents are evaluated in a cell in culture,e.g., a cell expressing a mutant ESR1 gene (e.g., a mammalian cell, atumor cell or cell line, a recombinant cell). In yet other embodiments,the anti-cancer agents are evaluated cell in vivo (a mutantESR1-expressing cell present in a subject, e.g., an animal subject(e.g., an in vivo animal model).

Exemplary parameters evaluated include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to a mutant ESR1 polypeptide; a binding competition between aknown ligand (e.g., estrogen or an estrogen inhibitor, such astamoxifen) and the candidate agent to a mutant ESR1 polypeptide;

(ii) a change in estrogen receptor activity, e.g., binding of estrogento the mutant ESR1 receptor polypeptide (e.g., an increased or decreasedbinding of estrogen); or a change in Activation Function activity, e.g.,decreased or increased transcription activation activity; or a change inDNA binding activity;

(iii) a change in an activity of a cell containing a mutant ESR1 (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology or tumorigenicity of the cell;

(iv) a change in tumor present in an animal subject, e.g., size,appearance, proliferation, of the tumor; or

-   -   (v) a change in the level, e.g., expression level, of a mutant        ESR1 polypeptide or nucleic acid molecule.

In one embodiment, a change in a cell free assay in the presence of acandidate agent is evaluated. For example, an activity of a mutant ESR1polypeptide, or interaction of a mutant ESR1 polypeptide with a ligand(e.g., estrogen or tamoxifen, or a DNA target sequence) can be detected.

In other embodiments, a change in an activity of a cell is detected in acell in culture, e.g., a cell expressing a mutant ESR1 polypeptide(e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell).In one embodiment, the cell is a recombinant cell that is modified toexpress a mutant ESR1 nucleic acid, e.g., is a recombinant celltransfected with a mutant ESR1 nucleic acid. The transfected cell canshow a change in response to the expressed mutant ESR1, e.g., increasedproliferation, changes in morphology, increased tumorigenicity, and/oracquired a transformed phenotype. A change in any of the activities ofthe cell, e.g., the recombinant cell, in the presence of the candidateagent can be detected. For example, a decrease in one or more of:proliferation, tumorigenicity, or transformed morphology, in thepresence of the candidate agent can be indicative of an inhibitor of amutant ESR1. In other embodiments, a change in binding activity orphosphorylation as described herein is detected.

In yet other embodiments, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,the animal model is a tumor containing animal or a xenograft comprisingcells expressing a mutant ESR1 (e.g., tumorigenic cells expressing amutant ESR1). The anti-cancer agents can be administered to the animalsubject and a change in the tumor is detected. In one embodiment, thechange in the tumor includes one or more of a tumor growth, tumor size,tumor burden, survival, is evaluated. A decrease in one or more of tumorgrowth, tumor size, tumor burden, or an increased survival is indicativethat the candidate agent is an inhibitor.

The screening methods and assays are described in more detail hereinbelow.

Screening Methods

In another aspect, the invention features a method, or assay, forscreening for agents that modulate, e.g., inhibit, the expression oractivity of a mutant ESR1, e.g., a mutant ESR1 as described herein. Themethod includes contacting a mutant ESR1 polypeptide, or a cellexpressing a mutant ESR1, with a candidate agent; and detecting a changein a parameter associated with the mutant ESR1, e.g., a change in theexpression or an activity of the mutant ESR1. The method can,optionally, include comparing the treated parameter to a referencevalue, e.g., a control sample (e.g., comparing a parameter obtained froma sample with the candidate agent to a parameter obtained from a samplewithout the candidate agent). In one embodiment, if a decrease inexpression or activity of the mutant ESR1 is detected, the candidateagent is identified as an inhibitor. In another embodiment, if anincrease in expression or activity of the mutant ESR1 is detected, thecandidate agent is identified as an activator. In certain embodiments,the mutant ESR1 is a nucleic acid molecule or a polypeptide as describedherein.

In one embodiment, the contacting step is effected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is effected in a cell in culture, e.g.,a cell expressing a mutant ESR1 (e.g., a mammalian cell, a tumor cell orcell line, a recombinant cell). In yet other embodiments, the contactingstep is effected in a cell in vivo (a mutant ESR1-expressing cellpresent in a subject, e.g., an animal subject (e.g., an in vivo animalmodel).

Exemplary parameters evaluated include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to a mutant ESR1 polypeptide; a binding competition between aknown ligand (e.g., estrogen, or an estrogen inhibitor, such astamoxifen) and the candidate agent to a mutant ESR1 polypeptide;

(ii) a change in transcriptional activation activity or DNA bindingactivity as measured, for example, by fusing an estrogen responseelement (ERE) to a reporter gene; DNA binding activity can also bemeasure by gel-shift assay;

(iii) a change in an activity of a cell containing a mutant ESR1 (e.g.,a tumor cell or a recombinant cell), e.g., a change in proliferation,morphology or tumorigenicity of the cell;

(iv) a change in tumor present in an animal subject, e.g., size,appearance, proliferation, of the tumor; or

(v) a change in the level, e.g., expression level, of a mutant ESR1polypeptide or nucleic acid molecule.

In one embodiment, a change in a cell free assay in the presence of acandidate agent is evaluated. For example, an activity of a mutant ESR1polypeptide, or interaction of a mutant ESR1 polypeptide with a ligandcan be detected. In one embodiment, a mutant ESR1 polypeptide iscontacted with a ligand, e.g., in solution, and a candidate agent ismonitored for an ability to modulate, e.g., inhibit, an interaction,e.g., binding, between the mutant ESR1 polypeptide and the ligand. Inone exemplary assay, purified ESR1 protein is contacted with a ligand,e.g., in solution, and a candidate agent is monitored for an ability toinhibit interaction of the protein with the ligand, or to inhibitactivity (e.g., DNA binding activity) of the mutant ESR1 protein. Aneffect on an interaction between the mutant protein and a ligand can bemonitored by methods known in the art, such as by absorbance, and aneffect on ESR1 activity levels, e.g., DNA binding or transcriptionactivation activity of the mutant ESR1 can be assayed, e.g., bygel-shift assays, reporter gene assays, and other methods known in theart.

In other embodiments, a change in an activity of a cell is detected in acell in culture, e.g., a cell expressing a mutant ESR1 polypeptide(e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell).In one embodiment, the cell is a recombinant cell that is modified toexpress a mutant ESR1 nucleic acid, e.g., is a recombinant celltransfected with a mutant ESR1 nucleic acid. The transfected cell canshow a change in response to the expressed mutant ESR1, e.g., increasedproliferation, changes in morphology, increased tumorigenicity, and/oracquired a transformed phenotype. A change in any of the activities ofthe cell, e.g., the recombinant cell, in the presence of the candidateagent can be detected. For example, a decrease in one or more of:proliferation, tumorigenicity, transformed morphology, in the presenceof the candidate agent can be indicative of an inhibitor of a mutantESR1. In other embodiments, a change in binding activity orphosphorylation as described herein is detected.

In an exemplary cell-based assay, a nucleic acid comprising a mutantESR1 can be expressed in a cell, such as a cell (e.g., a mammalian cell)in culture. The cell containing a nucleic acid expressing the mutantESR1 can be contacted with a candidate agent, and the cell is monitoredfor an effect of the candidate agent. A candidate agent that causesdecreased cell proliferation or cell death can be determined to be acandidate for treating a tumor (e.g., a cancer) that carries a mutantESR1.

In one embodiment, a cell containing a nucleic acid expressing a mutantESR1 can be monitored for expression of the mutant ESR1 protein. Proteinexpression can be monitored by methods known in the art, such as by,e.g., mass spectrometry (e.g., tandem mass spectrometry), a reporterassay (e.g., a fluorescence-based assay), Western blot, andimmunohistochemistry. By one method, decreased mutant ESR1 expression isdetected. A candidate agent that causes decreased expression of themutant ESR1 protein as compared to a cell that does not contain themutant ESR1 nucleic acid can be determined to be a candidate fortreating a tumor (e.g., a cancer) that carries a mutant ESR1.

A cell containing a nucleic acid expressing a mutant ESR1 can bemonitored for altered DNA binding or transcriptional activationactivity. Transcriptional activation activity can be assayed bymeasuring the effect of a candidate agent on expression of a reportergene under control of a ERE (estrogen response element). An ERE has theconsensus sequence GGTCANNNTGACC (SEQ ID NO: 5) (Klein-Hitpass et al.,Cell 46:1053-1061, 1986).

In yet other embodiments, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,the animal model is a tumor containing animal or a xenograft comprisingcells expressing a mutant ESR1 (e.g., tumorigenic cells expressing aESR1 that carries a mutation in the ligand binding domain). Thecandidate agent can be administered to the animal subject and a changein the tumor is detected. In one embodiment, the change in the tumorincludes one or more of a tumor growth, tumor size, tumor burden, orsurvival. A decrease in one or more of tumor growth, tumor size, tumorburden, or an increased survival is indicative that the candidate agentis an inhibitor.

In one exemplary animal model, a xenograft is created by injecting cellsinto a mouse. A candidate agent is administered to the mouse, e.g., byinjection (such as subcutaneous, intraperitoneal, or tail veininjection, or by injection directly into the tumor) or oral delivery,and the tumor is observed to determine an effect of the candidateanti-cancer agent. The health of the animal is also monitored, such asto determine if an animal treated with a candidate agent surviveslonger. A candidate agent that causes growth of the tumor to slow orstop, or causes the tumor to shrink in size, or causes decreased tumorburden, or increases survival time, can be considered to be a candidatefor treating a tumor (e.g., a cancer) that carries an ESR1 mutation.

In another exemplary animal assay, cells expressing a mutant ESR1 areinjected into the tail vein, e.g., of a mouse, to induce metastasis. Acandidate agent is administered to the mouse, e.g., by injection (suchas subcutaneous, intraperitoneal, or tail vein injection, or byinjection directly into the tumor) or oral delivery, and the tumor isobserved to determine an effect of the candidate anti-cancer agent. Acandidate agent that inhibits or prevents or reduces metastasis, orincreases survival time, can be considered to be a candidate fortreating a tumor (e.g., a cancer) that carries a mutant ESR1.

Cell proliferation can be measured by methods known in the art, such asPCNA (Proliferating cell nuclear antigen) assay, 5-bromodeoxyuridine(BrdUrd) incorporation, Ki-67 assay, mitochondrial respiration, orpropidium iodide staining. Cells can also be measured for apoptosis,such as by use of a TUNEL (Terminal Deoxynucleotide Transferase dUTPNick End Labeling) assay. Cells can also be assayed for presence ofangiogenesis using methods known in the art, such as by measuringendothelial tube formation or by measuring the growth of blood vesselsfrom subcutaneous tissue, such as into a solid gel of basement membrane.

In other embodiments, a change in expression of a mutant ESR1 can bemonitored by detecting the nucleic acid or protein levels, e.g., usingthe methods described herein.

In certain embodiments, the screening methods described herein can berepeated and/or combined. In one embodiment, a candidate agent that isevaluated in a cell-free or cell-based assay described herein can befurther tested in an animal subject.

In one embodiment, the candidate agent is identified and re-tested inthe same or a different assay. For example, a test compound isidentified in an in vitro or cell-free system, and re-tested in ananimal model or a cell-based assay. Any order or combination of assayscan be used. For example, a high throughput assay can be used incombination with an animal model or tissue culture.

Candidate agents suitable for use in the screening assays describedherein include, e.g., small molecule compounds, nucleic acids (e.g.,siRNA, aptamers, short hairpin RNAs, antisense oligonucleotides,ribozymes, antagomirs, microRNA mimics or DNA, e.g., for gene therapy)or polypeptides, e.g., antibodies (e.g., full length antibodies orantigen-binding fragments thereof, Fab fragments, or scFv fragments).The candidate anti-cancer agents can be obtained from a library (e.g., acommercial library), or can be rationally designed, such as to target anactive site in a functional domain of ESR1 (e.g., the ligand bindingdomain of ESR1).

In other embodiments, the method, or assay, includes providing a stepbased on proximity-dependent signal generation, e.g., a two-hybrid assaythat includes a first mutant protein (e.g., a mutant ESR1 protein), anda second protein (e.g., a ligand), contacting the two-hybrid assay witha test compound, under conditions wherein said two hybrid assay detectsa change in the formation and/or stability of the complex, e.g., theformation of the complex initiates transcription activation of areporter gene.

In one non-limiting example, the three-dimensional structure of theligand binding domain of the mutant ESR1 is determined by crystallizingthe complex formed by the mutant ESR1 and a known inhibitor. Rationaldrug design is then used to identify new test agents by makingalterations in the structure of a known inhibitor or by designing smallmolecule compounds that bind to the ligand binding domain of the ESR1polypeptide.

The candidate agents can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; peptoid libraries (libraries of molecules havingthe functionalities of peptides, but with a novel, non-peptide backbonewhich are resistant to enzymatic degradation but which neverthelessremain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med.Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means known in the art (e.g., using afluorimeter).

In another embodiment, determining the ability of the mutant ESR1protein to bind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

Nucleic Acid Inhibitors

In yet another embodiment, the mutant ESR1 inhibitor inhibits theexpression of nucleic acid encoding a mutant ESR1. Examples of suchmutant ESR1 inhibitors include nucleic acid molecules, for example,antisense molecules, ribozymes, siRNA, triple helix molecules thathybridize to a nucleic acid encoding a mutant ESR1, or a transcriptionregulatory region, and blocks or reduces mRNA expression of the mutantESR1.

In one embodiment, the nucleic acid antagonist is a siRNA that targetsmRNA encoding mutant ESR1. Other types of antagonistic nucleic acids canalso be used, e.g., a dsRNA, a ribozyme, a triple-helix former, or anantisense nucleic acid. Accordingly, isolated nucleic acid moleculesthat are nucleic acid inhibitors, e.g., antisense, RNAi, to a mutantESR1-encoding nucleic acid molecule are provided.

An “antisense” nucleic acid can include a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. The antisense nucleic acid can becomplementary to an entire mutant ESR1 coding strand, or to only aportion thereof. For example, the antisense nucleic acid can becomplementary to the sequence in the ligand-binding domain that carriesthe mutation, e.g., can be complementary to the fusion junction in theligand-binding domain created by the six-nucleotide deletion describedherein. In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding a mutant ESR1 (e.g., the 5′ and 3′ untranslatedregions). Anti-sense agents can include, for example, from about 8 toabout 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g.,about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases.Anti-sense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA can interfere withone or more of the normal functions of mRNA. The functions of mRNA to beinterfered with include all key functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding mutant ESR1. The complementary region can extend for betweenabout 8 to about 80 nucleobases. The compounds can include one or moremodified nucleobases. Modified nucleobases are known in the art.Descriptions of modified nucleic acid agents are also available. See,e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and 5,093,246; Woolf et al.(1992) Proc Natl Acad Sci USA; Antisense RNA and DNA, D. A. Melton, Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988);89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C.(1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher (1992) Bioassays 14:807-15.

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a mutant ESR1 to thereby inhibitexpression of the protein, e.g., by inhibiting transcription and/ortranslation. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies which bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are typical.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

siRNAs are small double stranded RNAs (dsRNAs) that optionally includeoverhangs. For example, the duplex region of an siRNA is about 18 to 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotidesin length. Typically, the siRNA sequences are exactly complementary tothe target mRNA. dsRNAs and siRNAs in particular can be used to silencegene expression in mammalian cells (e.g., human cells). siRNAs alsoinclude short hairpin RNAs (shRNAs) with 29-base-pair stems and2-nucleotide 3′ overhangs. See, e.g., Clemens et al. (2000) Proc. Natl.Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et al.(2002) Proc. Natl. Acad. Sci. USA 99:9942-9947; Siolas et al. (2005),Nat. Biotechnol. 23(2):227-31; 20040086884; U.S. 20030166282;20030143204; 20040038278; and 20030224432.

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. A ribozyme having specificity for a mutant ESR1-encodingnucleic acid can include one or more sequences complementary to thenucleotide sequence of a mutant ESR1 cDNA disclosed herein (i.e., SEQ IDNO:3), and a sequence having known catalytic sequence responsible formRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach(1988) Nature 334:585-591). For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina mutant ESR1-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,mutant ESR1 mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Mutant ESR1 gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of the mutant ESR1 toform triple helical structures that prevent transcription of the mutantESR1 gene in target cells. See generally, Helene, C. (1991) AnticancerDrug Des. 6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14:807-15. The potential sequencesthat can be targeted for triple helix formation can be increased bycreating a so-called “switchback” nucleic acid molecules. Switchbackmolecules are synthesized in an alternating 5′-3′, 3′-5′ manner, suchthat they base pair with first one strand of a duplex and then theother, eliminating the necessity for a sizeable stretch of eitherpurines or pyrimidines to be present on one strand of a duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

A mutant ESR1 nucleic acid molecule can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For non-limiting examplesof synthetic oligonucleotides with modifications see Toulmé (2001)Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44.Such phosphoramidite oligonucleotides can be effective antisense agents.

For example, the deoxyribose phosphate backbone of the nucleic acidmolecules can be modified to generate peptide nucleic acids (see HyrupB. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As usedherein, the terms “peptide nucleic acid” or “PNA” refers to a nucleicacid mimic, e.g., a DNA mimic, in which the deoxyribose phosphatebackbone is replaced by a pseudopeptide backbone and only the fournatural nucleobases are retained. The neutral backbone of a PNA canallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in HyrupB. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci.93: 14670-675.

PNAs of mutant ESR1 nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of mutant ESR1 nucleic acid molecules canalso be used in the analysis of single base pair mutations in a gene(e.g., by PNA-directed PCR clamping); as ‘artificial restrictionenzymes’ when used in combination with other enzymes (e.g., 51 nucleases(Hyrup B. et al. (1996) supra)); or as probes or primers for DNAsequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefesupra).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652;WO88/09810) or the blood-brain barrier (see, e.g., WO 89/10134). Inaddition, oligonucleotides can be modified with hybridization-triggeredcleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976)or intercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549).To this end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

Evaluation of Subjects

Subjects, e.g., patients, can be evaluated for the presence of a mutantESR1. A patient can be evaluated, for example, by determining thegenomic sequence of the patient, e.g., by an NGS method. Alternatively,or in addition, evaluation of a patient can include directly assayingfor the presence of a mutant ESR1 in the patient, such as by an assay todetect a mutant ESR1 nucleic acid (e.g., DNA or RNA), such as by,Southern blot, Northern blot, or RT-PCR, e.g., qRT-PCR. Alternatively,or in addition, a patient can be evaluated for the presence of a mutantESR1 protein, such as by immunohistochemistry, Western blot,immunoprecipitation, or immunomagnetic bead assay.

Evaluation of a patient can also include a cytogenetic assay, such as byfluorescence in situ hybridization (FISH), to identify the chromosomalrearrangement resulting in the mutant ESR1. For example, to performFISH, at least a first probe tagged with a first detectable label can bedesigned to target a sequence in the ligand binding domain of ESR1,e.g., in one or more of exons 8-12 of KIF5B (see FIG. 1).

Additional methods for mutant ESR1 detection are provided below.

In one aspect, the results of a clinical trial, e.g., a successful orunsuccessful clinical trial, can be repurposed to identify agents thattarget a mutant ESR1. By one exemplary method, a candidate agent used ina clinical trial can be reevaluated to determine if the agent in thetrial targets a mutant ESR1, or is effective to treat a tumor containinga mutant ESR1. For example, subjects who participated in a clinicaltrial for an agent, such as an estrogen inhibitor, can be identified.Patients who experienced an improvement in symptoms, e.g., cancer (e.g.,breast cancer) symptoms, such as decreased tumor size, or decreased rateof tumor growth, can be evaluated for the presence of a mutation in theligand binding domain of ESR1. Patients who did not experience animprovement in cancer symptoms can also be evaluated for the presence ofan ESR1 mutation. Where patients carrying a mutation in the ligandbinding domain of ESR1 are found to have been more likely to respond tothe test agent than patients who did not carry a mutation in the ligandbinding domain, then the agent is determined to be an appropriatetreatment option for a patient carrying the mutant ESR1.

“Reevaluation” of patients can include, for example, determining thegenomic sequence of the patients, or a subset of the clinical trialpatients, e.g., by an NGS method. Alternatively, or in addition,reevaluation of the patients can include directly assaying for thepresence of an ESR1 mutation in the patient, such as by an assay todetect a mutant ESR1 nucleic acid (e.g., RNA), such as by RT-PCR, e.g.,qRT-PCR. Alternatively, or in addition, a patient can be evaluated forthe presence of a mutant ESR1 protein, such as by immunohistochemistry,Western blot, immunoprecipitation, or immunomagnetic bead assay.

Clinical trials suitable for repurposing as described above includetrials that tested estrogen receptor inhibitors, or estrogen mimics,SERMs or aromatase inhibitors.

Methods for Detection of Mutant ESR1 Nucleic Acids and Polypeptides

Methods for evaluating an ESR1 gene, mutations and/or gene products areknown to those of skill in the art. In one embodiment, the mutant ESR1is detected in a nucleic acid molecule by a method chosen from one ormore of: nucleic acid hybridization assays, amplification-based assays(e.g., polymerase chain reaction (PCR)), PCR-RFLP assay, real-time PCR,sequencing, screening analysis (including metaphase cytogenetic analysisby standard karyotype methods, FISH (e.g., break away FISH), spectralkaryotyping or MFISH, comparative genomic hybridization), in situhybridization, SSP, HPLC or mass-spectrometric genotyping.

In certain embodiments, the evaluation methods include theprobes/primers described herein.

In one embodiment, probes/primers can be designed to detect a mutationin ESR1. The ESR1 probes/primers can be from nucleotides 1-1788 of SEQID NO:1, or from nucleotides 1-1782 of SEQ ID NO:3 (e.g., can hybridizeto the nucleotides encoding exons 5b-12 of the ESR1 protein). In someembodiments, the ESR1 probes/primers can be from nucleotides 934-1638 ofSEQ ID NO:1 or nucleotides 934-1632 of SEQ ID NO:3 (e.g., can hybridizeto the nucleotides encoding exons 8-12 of the ESR1 protein). Theseprobes/primers are suitable, e.g., for PCR amplification. For PCR, e.g.,to amply the region including the ligand binding domain of ESR1, forwardprimers can be designed to hybridize to ESR1 sequence from nucleotide761 of SEQ ID NO:1, and reverse primers can be designed to hybridizefrom nucleotide 2158 of SEQ ID NO:1.

In one embodiment, amplification-based assays can be used to measurepresence/absence and copy number. In such amplification-based assays,the nucleic acid sequences act as a template in an amplificationreaction (e.g., Polymerase Chain Reaction (PCR). In a quantitativeamplification, the amount of amplification product will be proportionalto the amount of template in the original sample. Comparison toappropriate controls, e.g., healthy tissue, provides a measure of thecopy number.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that can be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis, et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR can also be used in the methods of theinvention. In fluorogenic quantitative PCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan® and SYBR® green.

In one embodiment, a TaqMan® assay is used to identify a deletion in theER gene, e.g., a 6 nucleotide deletion as described herein, such as byutilizing a probe that binds specifically to the fusion junction createdby the deletion, and a control probe that binds to the wildtypesequence, and probes that bind outside of the mutated sequence for PCRamplification.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Chromosomal probes are typically about 50 to about 10⁵ nucleotides inlength. Longer probes typically comprise smaller fragments of about 100to about 500 nucleotides in length. Probes that hybridize withcentromeric DNA and locus-specific DNA are available commercially, forexample, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc.(Eugene, Oreg.) or from Cytocell (Oxfordshire, UK). Alternatively,probes can be made non-commercially from chromosomal or genomic DNAthrough standard techniques. For example, sources of DNA that can beused include genomic DNA, cloned DNA sequences, somatic cell hybridsthat contain one, or a part of one, chromosome (e.g., human chromsome)along with the normal chromosome complement of the host, and chromosomespurified by flow cytometry or microdis section. The region of interestcan be isolated through cloning, or by site-specific amplification viathe polymerase chain reaction (PCR). See, for example, Nath and Johnson,Biotechnic Histochem., 1998, 73(1):6-22, Wheeless et al., Cytometry1994, 17:319-326, and U.S. Pat. No. 5,491,224.

Additional exemplary methods include, traditional “direct probe” methodssuch as Southern blots or in situ hybridization (e.g., fluorescence insitu hybridization (FISH) and FISH plus SKY), and “comparative probe”methods such as comparative genomic hybridization (CGH), e.g.,cDNA-based or oligonucleotide-based CGH, can be used. The methods can beused in a wide variety of formats including, but not limited to,substrate (e.g., membrane or glass) bound methods or array-basedapproaches.

Additional protocols for FISH detection are described below.

The probes to be used hybridize to a specific region of a chromosome todetermine whether a cytogenetic abnormality is present in this region.One type of cytogenetic abnormality is a deletion. Although deletionscan be of one or more entire chromosomes, deletions normally involveloss of part of one or more chromosomes. If the entire region of achromosome that is contained in a probe is deleted from a cell,hybridization of that probe to the DNA from the cell will normally notoccur and no signal will be present on that chromosome. If the region ofa chromosome that is partially contained within a probe is deleted froma cell, hybridization of that probe to the DNA from the cell can stilloccur, but less of a signal can be present. For example, the loss of asignal is compared to probe hybridization to DNA from control cells thatdo not contain the genetic abnormalities which the probes are intendedto detect. In some embodiments, at least 1, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, ormore cells are enumerated for presence of the cytogenetic abnormality.

Cytogenetic abnormalities to be detected can include, but are notlimited to, non-reciprocal translocations, intra-chromosomal inversions,point mutations, deletions, gene copy number changes, gene expressionlevel changes, and germ line mutations. In particular, one type ofcytogenetic abnormality is a duplication. Duplications can be of entirechromosomes, or of regions smaller than an entire chromosome. If theregion of a chromosome that is contained in a probe is duplicated in acell, hybridization of that probe to the DNA from the cell will normallyproduce at least one additional signal as compared to the number ofsignals present in control cells with no abnormality of the chromosomalregion contained in the probe. Chromosomal probes are labeled so thatthe chromosomal region to which they hybridize can be detected. Probestypically are directly labeled with a fluorophore, an organic moleculethat fluoresces after absorbing light of lower wavelength/higher energy.The fluorophore allows the probe to be visualized without a secondarydetection molecule. After covalently attaching a fluorophore to anucleotide, the nucleotide can be directly incorporated into the probewith standard techniques such as nick translation, random priming, andPCR labeling. Alternatively, deoxycytidine nucleotides within the probecan be transaminated with a linker. The fluorophore then is covalentlyattached to the transaminated deoxycytidine nucleotides. See, U.S. Pat.No. 5,491,224.

U.S. Pat. No. 5,491,224 describes probe labeling as a number of thecytosine residues having a fluorescent label covalently bonded thereto.The number of fluorescently labeled cytosine bases is sufficient togenerate a detectable fluorescent signal while the individual so labeledDNA segments essentially retain their specific complementary binding(hybridizing) properties with respect to the chromosome or chromosomeregion to be detected. Such probes are made by taking the unlabeled DNAprobe segment, transaminating with a linking group a number ofdeoxycytidine nucleotides in the segment, covalently bonding afluorescent label to at least a portion of the transaminateddeoxycytidine bases.

Probes can also be labeled by nick translation, random primer labelingor PCR labeling. Labeling is done using either fluorescent (direct)- orhaptene (indirect)-labeled nucleotides. Representative, non-limitingexamples of labels include: AMCA-6-dUTP, CascadeBlue-4-dUTP,Fluorescein-12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP,Cy5-dUTP, Biotin(BIO)-11-dUTP, Digoxygenin(DIG)-11-dUTP or Dinitrophenyl(DNP)-11-dUTP.

Probes also can be indirectly labeled with biotin or digoxygenin, orlabeled with radioactive isotopes such as ³²P and ³H, although secondarydetection molecules or further processing then is required to visualizethe probes. For example, a probe labeled with biotin can be detected byavidin conjugated to a detectable marker. For example, avidin can beconjugated to an enzymatic marker such as alkaline phosphatase orhorseradish peroxidase. Enzymatic markers can be detected in standardcolorimetric reactions using a substrate and/or a catalyst for theenzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Probes can also be prepared such that a fluorescent or other label isnot part of the DNA before or during the hybridization, and is addedafter hybridization to detect the probe hybridized to a chromosome. Forexample, probes can be used that have antigenic molecules incorporatedinto the DNA. After hybridization, these antigenic molecules aredetected using specific antibodies reactive with the antigenicmolecules. Such antibodies can themselves incorporate a fluorochrome, orcan be detected using a second antibody with a bound fluorochrome.

However treated or modified, the probe DNA is commonly purified in orderto remove unreacted, residual products (e.g., fluorochrome molecules notincorporated into the DNA) before use in hybridization.

Prior to hybridization, chromosomal probes are denatured according tomethods well known in the art. Probes can be hybridized or annealed tothe chromosomal DNA under hybridizing conditions. “Hybridizingconditions” are conditions that facilitate annealing between a probe andtarget chromosomal DNA. Since annealing of different probes will varydepending on probe length, base concentration and the like, annealing isfacilitated by varying probe concentration, hybridization temperature,salt concentration and other factors well known in the art.

Hybridization conditions are facilitated by varying the concentrations,base compositions, complexities, and lengths of the probes, as well assalt concentrations, temperatures, and length of incubation. Forexample, in situ hybridizations are typically performed in hybridizationbuffer containing 1-2×SSC, 50-65% formamide and blocking DNA to suppressnon-specific hybridization. In general, hybridization conditions, asdescribed above, include temperatures of about 25° C. to about 55° C.,and incubation lengths of about 0.5 hours to about 96 hours.

Non-specific binding of chromosomal probes to DNA outside of the targetregion can be removed by a series of washes. Temperature andconcentration of salt in each wash are varied to control stringency ofthe washes. For example, for high stringency conditions, washes can becarried out at about 65° C. to about 80° C., using 0.2× to about 2×SSC,and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40(NP40). Stringency can be lowered by decreasing the temperature of thewashes or by increasing the concentration of salt in the washes. In someapplications it is necessary to block the hybridization capacity ofrepetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-I DNA is used to block non-specific hybridization. Afterwashing, the slide is allowed to drain and air dry, then mountingmedium, a counterstain such as DAPI, and a coverslip are applied to theslide. Slides can be viewed immediately or stored at −20° C. beforeexamination.

For fluorescent probes used in fluorescence in situ hybridization (FISH)techniques, fluorescence can be viewed with a fluorescence microscopeequipped with an appropriate filter for each fluorophore, or by usingdual or triple band-pass filter sets to observe multiple fluorophores.See, for example, U.S. Pat. No. 5,776,688. Alternatively, techniquessuch as flow cytometry can be used to examine the hybridization patternof the chromosomal probes.

In CGH methods, a first collection of nucleic acids (e.g., from asample, e.g., a possible tumor) is labeled with a first label, while asecond collection of nucleic acids (e.g., a control, e.g., from ahealthy cell/tissue) is labeled with a second label. The ratio ofhybridization of the nucleic acids is determined by the ratio of the two(first and second) labels binding to each fiber in the array. Wherethere are chromosomal deletions or multiplications, differences in theratio of the signals from the two labels will be detected and the ratiowill provide a measure of the copy number. Array-based CGH can also beperformed with single-color labeling (as opposed to labeling the controland the possible tumor sample with two different dyes and mixing themprior to hybridization, which will yield a ratio due to competitivehybridization of probes on the arrays). In single color CGH, the controlis labeled and hybridized to one array and absolute signals are read,and the possible tumor sample is labeled and hybridized to a secondarray (with identical content) and absolute signals are read. Copynumber difference is calculated based on absolute signals from the twoarrays. Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430,402; Methods in Molecular Biology, Vol. 33: In situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In oneembodiment, the hybridization protocol of Pinkel, et al. (1998) NatureGenetics 20: 207-211, or of Kallioniemi (1992) Proc. Natl Acad Sci USA89:5321-5325 (1992) is used. Array-based CGH is described in U.S. Pat.No. 6,455,258, the contents of each of which are incorporated herein byreference.

Nucleic Acid Samples

A variety of tissue samples can be the source of the nucleic acidsamples used in the present methods. Genomic or subgenomic DNA fragmentscan be isolated from a subject's sample (e.g., a tumor sample, a normaladjacent tissue (NAT), a blood sample or any normal control)). Incertain embodiments, the tissue sample is preserved as a frozen sampleor as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE)tissue preparation. For example, the sample can be embedded in a matrix,e.g., an FFPE block or a frozen sample. The isolating step can includeflow-sorting of individual chromosomes; and/or micro-dissecting asubject's sample (e.g., a tumor sample, a NAT, a blood sample).

Protocols for DNA isolation from a tissue sample are known in the art.Additional methods to isolate nucleic acids (e.g., DNA) fromformaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE)tissues are disclosed, e.g., in Cronin M. et al., (2004) Am J Pathol.164(1):35-42; Masuda N. et al., (1999) Nucleic Acids Res.27(22):4436-4443; Specht K. et al., (2001) Am J Pathol. 158(2):419-429,Ambion RecoverAll™ Total Nucleic Acid Isolation Protocol (Ambion, Cat.No. AM1975, September 2008), and QIAamp® DNA FFPE Tissue Handbook(Qiagen, Cat. No. 37625, October 2007). RecoverAll™ Total Nucleic AcidIsolation Kit uses xylene at elevated temperatures to solubilizeparaffin-embedded samples and a glass-fiber filter to capture nucleicacids. QIAamp® DNA FFPE Tissue Kit uses QIAamp® DNA Micro technology forpurification of genomic and mitochondrial DNA.

The isolated nucleic acid samples (e.g., genomic DNA samples) can befragmented or sheared by practicing routine techniques. For example,genomic DNA can be fragmented by physical shearing methods, enzymaticcleavage methods, chemical cleavage methods, and other methods wellknown to those skilled in the art. The nucleic acid library can containall or substantially all of the complexity of the genome. The term“substantially all” in this context refers to the possibility that therecan in practice be some unwanted loss of genome complexity during theinitial steps of the procedure. The methods described herein also areuseful in cases where the nucleic acid library is a portion of thegenome, i.e., where the complexity of the genome is reduced by design.In some embodiments, any selected portion of the genome can be used withthe methods described herein. In certain embodiments, the entire exomeor a subset thereof is isolated.

Methods can further include isolating a nucleic acid sample to provide alibrary (e.g., a nucleic acid library). In certain embodiments, thenucleic acid sample includes whole genomic, subgenomic fragments, orboth. The isolated nucleic acid samples can be used to prepare nucleicacid libraries. Thus, in one embodiment, the methods featured in theinvention further include isolating a nucleic acid sample to provide alibrary (e.g., a nucleic acid library as described herein). Protocolsfor isolating and preparing libraries from whole genomic or subgenomicfragments are known in the art (e.g., Illumina's genomic DNA samplepreparation kit). In certain embodiments, the genomic or subgenomic DNAfragment is isolated from a subject's sample (e.g., a tumor sample, anormal adjacent tissue (NAT), a blood sample or any normal control)). Inone embodiment, the sample (e.g., the tumor or NAT sample) is apreserved. For example, the sample is embedded in a matrix, e.g., anFFPE block or a frozen sample. In certain embodiments, the isolatingstep includes flow-sorting of individual chromosomes; and/ormicrodissecting a subject's sample (e.g., a tumor sample, a NAT, a bloodsample). In certain embodiments, the nucleic acid sample used togenerate the nucleic acid library is less than 5, less than 1 microgram,less than 500 ng, less than 200 ng, less than 100 ng, less than 50 ng orless than 20 ng (e.g., 10 ng or less).

In still other embodiments, the nucleic acid sample used to generate thelibrary includes RNA or cDNA derived from RNA. In some embodiments, theRNA includes total cellular RNA. In other embodiments, certain abundantRNA sequences (e.g., ribosomal RNAs) have been depleted. In someembodiments, the poly(A)-tailed mRNA fraction in the total RNApreparation has been enriched. In some embodiments, the cDNA is producedby random-primed cDNA synthesis methods. In other embodiments, the cDNAsynthesis is initiated at the poly(A) tail of mature mRNAs by priming byoligo(dT)-containing oligonucleotides. Methods for depletion, poly(A)enrichment, and cDNA synthesis are well known to those skilled in theart.

The method can further include amplifying the nucleic acid sample (e.g.,DNA or RNA sample) by specific or non-specific nucleic acidamplification methods that are well known to those skilled in the art.In some embodiments, certain embodiments, the nucleic acid sample isamplified, e.g., by whole-genome amplification methods such asrandom-primed strand-displacement amplification.

In other embodiments, the nucleic acid sample is fragmented or shearedby physical or enzymatic methods and ligated to synthetic adapters,size-selected (e.g., by preparative gel electrophoresis) and amplified(e.g., by PCR). In other embodiments, the fragmented and adapter-ligatedgroup of nucleic acids is used without explicit size selection oramplification prior to hybrid selection.

In other embodiments, the isolated DNA (e.g., the genomic DNA) isfragmented or sheared. In some embodiments, the library includes lessthan 50% of genomic DNA, such as a subfraction of genomic DNA that is areduced representation or a defined portion of a genome, e.g., that hasbeen subfractionated by other means. In other embodiments, the libraryincludes all or substantially all genomic DNA.

In some embodiments, the library includes less than 50% of genomic DNA,such as a subfraction of genomic DNA that is a reduced representation ora defined portion of a genome, e.g., that has been subfractionated byother means. In other embodiments, the library includes all orsubstantially all genomic DNA. Protocols for isolating and preparinglibraries from whole genomic or subgenomic fragments are known in theart (e.g., Illumina's genomic DNA sample preparation kit). AlternativeDNA shearing methods can be more automatable and/or more efficient(e.g., with degraded FFPE samples). Alternatives to DNA shearing methodscan also be used to avoid a ligation step during library preparation.

The methods described herein can be performed using a small amount ofnucleic acids, e.g., when the amount of source DNA is limiting (e.g.,even after whole-genome amplification). In one embodiment, the nucleicacid comprises less than about 5 μg, 4 μg, 3 μg, 2 μg, 1 μg, 0.8 μg, 0.7μg, 0.6 μg, 0.5 μg, or 400 ng, 300 ng, 200 ng, 100 ng, 50 ng, or 20 ngor less of nucleic acid sample. For example, to prepare 500 ng ofhybridization-ready nucleic acids, one typically begins with 3 μg ofgenomic DNA. One can start with less, however, if one amplifies thegenomic DNA (e.g., using PCR) before the step of solution hybridization.Thus it is possible, but not essential, to amplify the genomic DNAbefore solution hybridization.

In some embodiments, a library is generated using DNA (e.g., genomicDNA) from a sample tissue, and a corresponding library is generated withRNA (or cDNA) isolated from the same sample tissue.

Design of Baits

A bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule,which can hybridize to (e.g., be complementary to), and thereby allowcapture of a target nucleic acid. In one embodiment, a bait is an RNAmolecule. In other embodiments, a bait includes a binding entity, e.g.,an affinity tag, that allows capture and separation, e.g., by binding toa binding entity, of a hybrid formed by a bait and a nucleic acidhybridized to the bait. In one embodiment, a bait is suitable forsolution phase hybridization.

Baits can be produced and used by methods and hybridization conditionsas described in US 2010/0029498 and Gnirke, A. et al. (2009) NatBiotechnol. 27(2):182-189, and U.S. Ser. No. 61/428,568, filed Dec. 30,2010, incorporated herein by reference. For example, biotinylated RNAbaits can be produced by obtaining a pool of synthetic longoligonucleotides, originally synthesized on a microarray, and amplifyingthe oligonucleotides to produce the bait sequences. In some embodiments,the baits are produced by adding an RNA polymerase promoter sequence atone end of the bait sequences, and synthesizing RNA sequences using RNApolymerase. In one embodiment, libraries of syntheticoligodeoxynucleotides can be obtained from commercial suppliers, such asAgilent Technologies, Inc., and amplified using known nucleic acidamplification methods.

Each bait sequence can include a target-specific (e.g., amember-specific) bait sequence and universal tails on each end. As usedherein, the term “bait sequence” can refer to the target-specific baitsequence or the entire oligonucleotide including the target-specific“bait sequence” and other nucleotides of the oligonucleotide. In oneembodiment, a target-specific bait hybridizes to a nucleic acid sequencecomprising a nucleic acid sequence in exons 8, 9, 10, 11, or 12 of ESR1,e.g., in exon 8 of ESR1.

In one embodiment, the bait is an oligonucleotide about 200 nucleotidesin length, of which 170 nucleotides are target-specific “bait sequence.”The other 30 nucleotides (e.g., 15 nucleotides on each end) areuniversal arbitrary tails used for PCR amplification. The tails can beany sequence selected by the user. For example, the pool of syntheticoligonucleotides can include oligonucleotides of the sequence of5′-ATCGCACCAGCGTGTN₁₇₀CACTGCGGCTCCTCA-3′ with N₁₇₀ indicating thetarget-specific bait sequences.

The bait sequences described herein can be used for selection of exonsand short target sequences. In one embodiment, the bait is between about100 nucleotides and 300 nucleotides in length. In another embodiment,the bait is between about 130 nucleotides and 230 nucleotides in length.In yet another embodiment, the bait is between about 150 nucleotides and200 nucleotides in length. The target-specific sequences in the baits,e.g., for selection of exons and short target sequences, are betweenabout 40 nucleotides and 1000 nucleotides in length. In one embodiment,the target-specific sequence is between about 70 nucleotides and 300nucleotides in length. In another embodiment, the target-specificsequence is between about 100 nucleotides and 200 nucleotides in length.In yet another embodiment, the target-specific sequence is between about120 nucleotides and 170 nucleotides in length.

Sequencing

The invention also includes methods of sequencing nucleic acids. In oneembodiment, any of a variety of sequencing reactions known in the artcan be used to directly sequence at least a portion of a mutant ESR1. Inone embodiment, the mutant ESR1 sequence is compared to a correspondingreference (control) sequence.

In one embodiment, the sequence of the mutant ESR1 nucleic acid moleculeis determined by a method that includes one or more of: hybridizing anoligonucleotide, e.g., an allele specific oligonucleotide for onealteration described herein to said nucleic acid; hybridizing a primer,or a primer set (e.g., a primer pair), that amplifies a regioncomprising the mutation or a fusion junction of the allele; amplifying,e.g., specifically amplifying, a region comprising the mutation or afusion junction of the allele; attaching an adapter oligonucleotide toone end of a nucleic acid that comprises the mutation or a fusionjunction of the allele; generating an optical, e.g., a colorimetricsignal, specific to the presence of the one of the mutation or fusionjunction; hybridizing a nucleic acid comprising the mutation or fusionjunction to a second nucleic acid, e.g., a second nucleic acid attachedto a substrate; generating a signal, e.g., an electrical or fluorescentsignal, specific to the presence of the mutation or fusion junction; andincorporating a nucleotide into an oligonucleotide that is hybridized toa nucleic acid that contains the mutation or fusion junction.

In another embodiment, the sequence is determined by a method thatcomprises one or more of: determining the nucleotide sequence from anindividual nucleic acid molecule, e.g., where a signal corresponding tothe sequence is derived from a single molecule as opposed, e.g., from asum of signals from a plurality of clonally expanded molecules;determining the nucleotide sequence of clonally expanded proxies forindividual nucleic acid molecules; massively parallel short-readsequencing; template-based sequencing; pyrosequencing; real-timesequencing comprising imaging the continuous incorporation ofdye-labeling nucleotides during DNA synthesis; nanopore sequencing;sequencing by hybridization; nano-transistor array based sequencing;polony sequencing; scanning tunneling microscopy (STM) based sequencing;or nanowire-molecule sensor based sequencing.

Any method of sequencing known in the art can be used. Exemplarysequencing reactions include those based on techniques developed byMaxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger(Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety ofautomated sequencing procedures can be utilized when performing theassays (Biotechniques (1995) 19:448), including sequencing by massspectrometry (see, for example, U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/16101,entitled DNA Sequencing by Mass Spectrometry by H. Köster; U.S. Pat. No.5,547,835 and international patent application Publication Number WO94/21822 entitled DNA Sequencing by Mass Spectrometry Via ExonucleaseDegradation by H. Köster), and U.S. Pat. No. 5,605,798 and InternationalPatent Application No. PCT/US96/03651 entitled DNA Diagnostics Based onMass Spectrometry by H. Kôster; Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159).

Sequencing of nucleic acid molecules can also be carried out usingnext-generation sequencing (NGS). Next-generation sequencing includesany sequencing method that determines the nucleotide sequence of eitherindividual nucleic acid molecules or clonally expanded proxies forindividual nucleic acid molecules in a highly parallel fashion (e.g.,greater than 10⁵ molecules are sequenced simultaneously). In oneembodiment, the relative abundance of the nucleic acid species in thelibrary can be estimated by counting the relative number of occurrencesof their cognate sequences in the data generated by the sequencingexperiment. Next generation sequencing methods are known in the art, andare described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews11:31-46, incorporated herein by reference.

In one embodiment, the next-generation sequencing allows for thedetermination of the nucleotide sequence of an individual nucleic acidmolecule (e.g., Helicos BioSciences' HeliScope Gene Sequencing system,and Pacific Biosciences' PacBio RS system). In other embodiments, thesequencing method determines the nucleotide sequence of clonallyexpanded proxies for individual nucleic acid molecules (e.g., the Solexasequencer, Illumina Inc., San Diego, Calif.; 454 Life Sciences(Branford, Conn.), and Ion Torrent). e.g., massively parallel short-readsequencing (e.g., the Solexa sequencer, Illumina Inc., San Diego,Calif.), which generates more bases of sequence per sequencing unit thanother sequencing methods that generate fewer but longer reads. Othermethods or machines for next-generation sequencing include, but are notlimited to, the sequencers provided by 454 Life Sciences (Branford,Conn.), Applied Biosystems (Foster City, Calif.; SOLiD sequencer), andHelicos BioSciences Corporation (Cambridge, Mass.).

Platforms for next-generation sequencing include, but are not limitedto, Roche/454's Genome Sequencer (GS) FLX System, Illumina/Solexa'sGenome Analyzer (GA), Life/APG's Support Oligonucleotide LigationDetection (SOLiD) system, Polonator's G.007 system, Helicos BioSciences'HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RSsystem.

NGS technologies can include one or more of steps, e.g., templatepreparation, sequencing and imaging, and data analysis.

Template Preparation

Methods for template preparation can include steps such as randomlybreaking nucleic acids (e.g., genomic DNA or cDNA) into smaller sizesand generating sequencing templates (e.g., fragment templates ormate-pair templates). The spatially separated templates can be attachedor immobilized to a solid surface or support, allowing massive amountsof sequencing reactions to be performed simultaneously. Types oftemplates that can be used for NGS reactions include, e.g., clonallyamplified templates originating from single DNA molecules, and singleDNA molecule templates.

Methods for preparing clonally amplified templates include, e.g.,emulsion PCR (emPCR) and solid-phase amplification.

EmPCR can be used to prepare templates for NGS. Typically, a library ofnucleic acid fragments is generated, and adapters containing universalpriming sites are ligated to the ends of the fragment. The fragments arethen denatured into single strands and captured by beads. Each beadcaptures a single nucleic acid molecule. After amplification andenrichment of emPCR beads, a large amount of templates can be attachedor immobilized in a polyacrylamide gel on a standard microscope slide(e.g., Polonator), chemically crosslinked to an amino-coated glasssurface (e.g., Life/APG; Polonator), or deposited into individualPicoTiterPlate (PTP) wells (e.g., Roche/454), in which the NGS reactioncan be performed.

Solid-phase amplification can also be used to produce templates for NGS.Typically, forward and reverse primers are covalently attached to asolid support. The surface density of the amplified fragments is definedby the ratio of the primers to the templates on the support. Solid-phaseamplification can produce hundreds of millions spatially separatedtemplate clusters (e.g., Illumina/Solexa). The ends of the templateclusters can be hybridized to universal sequencing primers for NGSreactions.

Other methods for preparing clonally amplified templates also include,e.g., Multiple Displacement Amplification (MDA) (Lasken R. S. Curr OpinMicrobiol. 2007; 10(5):510-6). MDA is a non-PCR based DNA amplificationtechnique. The reaction involves annealing random hexamer primers to thetemplate and DNA synthesis by high fidelity enzyme, typically 029 at aconstant temperature. MDA can generate large sized products with lowererror frequency.

Template amplification methods such as PCR can be coupled with NGSplatforms to target or enrich specific regions of the genome (e.g.,exons). Exemplary template enrichment methods include, e.g.,microdroplet PCR technology (Tewhey R. et al., Nature Biotech. 2009,27:1025-1031), custom-designed oligonucleotide microarrays (e.g.,Roche/NimbleGen oligonucleotide microarrays), and solution-basedhybridization methods (e.g., molecular inversion probes (MIPs) (PorrecaG. J. et al., Nature Methods, 2007, 4:931-936; Krishnakumar S. et al.,Proc. Natl. Acad. Sci. USA, 2008, 105:9296-9310; Turner E. H. et al.,Nature Methods, 2009, 6:315-316), and biotinylated RNA capture sequences(Gnirke A. et al., Nat. Biotechnol. 2009; 27(2):182-9)

Single-molecule templates are another type of templates that can be usedfor NGS reaction. Spatially separated single molecule templates can beimmobilized on solid supports by various methods. In one approach,individual primer molecules are covalently attached to the solidsupport. Adapters are added to the templates and templates are thenhybridized to the immobilized primers. In another approach,single-molecule templates are covalently attached to the solid supportby priming and extending single-stranded, single-molecule templates fromimmobilized primers. Universal primers are then hybridized to thetemplates. In yet another approach, single polymerase molecules areattached to the solid support, to which primed templates are bound.

Sequencing and Imaging

Exemplary sequencing and imaging methods for NGS include, but are notlimited to, cyclic reversible termination (CRT), sequencing by ligation(SBL), single-molecule addition (pyrosequencing), and real-timesequencing.

CRT uses reversible terminators in a cyclic method that minimallyincludes the steps of nucleotide incorporation, fluorescence imaging,and cleavage. Typically, a DNA polymerase incorporates a singlefluorescently modified nucleotide corresponding to the complementarynucleotide of the template base to the primer. DNA synthesis isterminated after the addition of a single nucleotide and theunincorporated nucleotides are washed away. Imaging is performed todetermine the identity of the incorporated labeled nucleotide. Then inthe cleavage step, the terminating/inhibiting group and the fluorescentdye are removed. Exemplary NGS platforms using the CRT method include,but are not limited to, Illumina/Solexa Genome Analyzer (GA), which usesthe clonally amplified template method coupled with the four-color CRTmethod detected by total internal reflection fluorescence (TIRF); andHelicos BioSciences/HeliScope, which uses the single-molecule templatemethod coupled with the one-color CRT method detected by TIRF.

SBL uses DNA ligase and either one-base-encoded probes ortwo-base-encoded probes for sequencing. Typically, a fluorescentlylabeled probe is hybridized to its complementary sequence adjacent tothe primed template. DNA ligase is used to ligate the dye-labeled probeto the primer. Fluorescence imaging is performed to determine theidentity of the ligated probe after non-ligated probes are washed away.The fluorescent dye can be removed by using cleavable probes toregenerate a 5′-PO₄ group for subsequent ligation cycles. Alternatively,a new primer can be hybridized to the template after the old primer isremoved. Exemplary SBL platforms include, but are not limited to,Life/APG/SOLiD (support oligonucleotide ligation detection), which usestwo-base-encoded probes.

Pyrosequencing method is based on detecting the activity of DNApolymerase with another chemiluminescent enzyme. Typically, the methodallows sequencing of a single strand of DNA by synthesizing thecomplementary strand along it, one base pair at a time, and detectingwhich base was actually added at each step. The template DNA isimmobile, and solutions of A, C, G, and T nucleotides are sequentiallyadded and removed from the reaction. Light is produced only when thenucleotide solution complements the first unpaired base of the template.The sequence of solutions which produce chemiluminescent signals allowsthe determination of the sequence of the template. Exemplarypyrosequencing platforms include, but are not limited to, Roche/454,which uses DNA templates prepared by emPCR with 1-2 million beadsdeposited into PTP wells.

Real-time sequencing involves imaging the continuous incorporation ofdye-labeled nucleotides during DNA synthesis. Exemplary real-timesequencing platforms include, but are not limited to, PacificBiosciences platform, which uses DNA polymerase molecules attached tothe surface of individual zero-mode waveguide (ZMW) detectors to obtainsequence information when phospholinked nucleotides are beingincorporated into the growing primer strand; Life/VisiGen platform,which uses an engineered DNA polymerase with an attached fluorescent dyeto generate an enhanced signal after nucleotide incorporation byfluorescence resonance energy transfer (FRET); and LI-COR Biosciencesplatform, which uses dye-quencher nucleotides in the sequencingreaction.

Other sequencing methods for NGS include, but are not limited to,nanopore sequencing, sequencing by hybridization, nano-transistor arraybased sequencing, polony sequencing, scanning tunneling microscopy (STM)based sequencing, and nanowire-molecule sensor based sequencing.

Nanopore sequencing involves electrophoresis of nucleic acid moleculesin solution through a nano-scale pore which provides a highly confinedspace within which single-nucleic acid polymers can be analyzed.Exemplary methods of nanopore sequencing are described, e.g., in BrantonD. et al., Nat Biotechnol. 2008; 26(10):1146-53.

Sequencing by hybridization is a non-enzymatic method that uses a DNAmicroarray. Typically, a single pool of DNA is fluorescently labeled andhybridized to an array containing known sequences. Hybridization signalsfrom a given spot on the array can identify the DNA sequence. Thebinding of one strand of DNA to its complementary strand in the DNAdouble-helix is sensitive to even single-base mismatches when the hybridregion is short or is specialized mismatch detection proteins arepresent. Exemplary methods of sequencing by hybridization are described,e.g., in Hanna G. J. et al., J. Clin. Microbiol. 2000; 38 (7): 2715-21;and Edwards J. R. et al., Mut. Res. 2005; 573 (1-2): 3-12.

Polony sequencing is based on polony amplification andsequencing-by-synthesis via multiple single-base-extensions (FISSEQ).Polony amplification is a method to amplify DNA in situ on apolyacrylamide film. Exemplary polony sequencing methods are described,e.g., in US Patent Application Publication No. 2007/0087362.

Nano-transistor array based devices, such as Carbon NanoTube FieldEffect Transistor (CNTFET), can also be used for NGS. For example, DNAmolecules are stretched and driven over nanotubes by micro-fabricatedelectrodes. DNA molecules sequentially come into contact with the carbonnanotube surface, and the difference in current flow from each base isproduced due to charge transfer between the DNA molecule and thenanotubes. DNA is sequenced by recording these differences. ExemplaryNano-transistor array based sequencing methods are described, e.g., inU.S. Patent Application Publication No. 2006/0246497.

Scanning tunneling microscopy (STM) can also be used for NGS. STM uses apiezo-electric-controlled probe that performs a raster scan of aspecimen to form images of its surface. STM can be used to image thephysical properties of single DNA molecules, e.g., generating coherentelectron tunneling imaging and spectroscopy by integrating scanningtunneling microscope with an actuator-driven flexible gap. Exemplarysequencing methods using STM are described, e.g., in U.S. PatentApplication Publication No. 2007/0194225.

A molecular-analysis device which is comprised of a nanowire-moleculesensor can also be used for NGS. Such device can detect the interactionsof the nitrogenous material disposed on the nanowires and nucleic acidmolecules such as DNA. A molecule guide is configured for guiding amolecule near the molecule sensor, allowing an interaction andsubsequent detection. Exemplary sequencing methods usingnanowire-molecule sensor are described, e.g., in U.S. Patent ApplicationPublication No. 2006/0275779.

Double ended sequencing methods can be used for NGS. Double endedsequencing uses blocked and unblocked primers to sequence both the senseand antisense strands of DNA. Typically, these methods include the stepsof annealing an unblocked primer to a first strand of nucleic acid;annealing a second blocked primer to a second strand of nucleic acid;elongating the nucleic acid along the first strand with a polymerase;terminating the first sequencing primer; deblocking the second primer;and elongating the nucleic acid along the second strand. Exemplarydouble ended sequencing methods are described, e.g., in U.S. Pat. No.7,244,567.

Data Analysis

After NGS reads have been generated, they can be aligned to a knownreference sequence or assembled de novo.

For example, identifying genetic variations such as single-nucleotidepolymorphism and structural variants in a sample (e.g., a tumor sample)can be accomplished by aligning NGS reads to a reference sequence (e.g.,a wild-type sequence). Methods of sequence alignment for NGS aredescribed e.g., in Trapnell C. and Salzberg S. L. Nature Biotech., 2009,27:455-457.

Examples of de novo assemblies are described, e.g., in Warren R. et al.,Bioinformatics, 2007, 23:500-501; Butler J. et al., Genome Res., 2008,18:810-820; and Zerbino D. R. and Birney E., Genome Res., 2008,18:821-829.

Sequence alignment or assembly can be performed using read data from oneor more NGS platforms, e.g., mixing Roche/454 and Illumina/Solexa readdata.

Algorithms and methods for data analysis are described in U.S. Ser. No.61/428,568, filed Dec. 30, 2010, incorporated herein by reference.

Mutant ESR1 Expression Level

In certain embodiments, mutant ESR1 expression levels can also beassayed. Mutant ESR1 expression can be assessed by any of a wide varietyof methods for detecting expression of a transcribed molecule orprotein. Non-limiting examples of such methods include immunologicalmethods for detection of secreted, cell-surface, cytoplasmic, or nuclearproteins, protein purification methods, protein function or activityassays, nucleic acid hybridization methods, nucleic acid reversetranscription methods, and nucleic acid amplification methods.

In certain embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g., mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. Mutant ESR1 expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

Methods of detecting and/or quantifying the mutant ESR1 gene transcript(mRNA or cDNA made therefrom) using nucleic acid hybridizationtechniques are known to those of skill in the art (see Sambrook et al.supra). For example, one method for evaluating the presence, absence, orquantity of cDNA involves a Southern transfer as described above.Briefly, the mRNA is isolated (e.g., using an acidguanidinium-phenol-chloroform extraction method, Sambrook et al. supra.)and reverse transcribed to produce cDNA. The cDNA is then optionallydigested and run on a gel in buffer and transferred to membranes.Hybridization is then carried out using the nucleic acid probes specificfor the mutant ESR1 cDNA, e.g., using the probes and primers describedherein.

In other embodiments, mutant ESR1 expression is assessed by preparinggenomic DNA or mRNA/cDNA (i.e., a transcribed polynucleotide) from cellsin a subject sample, and by hybridizing the genomic DNA or mRNA/cDNAwith a reference polynucleotide which is a complement of apolynucleotide comprising the mutant ESR1, and fragments thereof. cDNAcan, optionally, be amplified using any of a variety of polymerase chainreaction methods prior to hybridization with the referencepolynucleotide. Expression of mutant ESR1 can likewise be detected usingquantitative PCR (QPCR) to assess the level of mutant ESR1 expression.

Detection of Mutant ESR1 Polypeptide

The activity or level of a mutant ESR1 polypeptide can also be detectedand/or quantified by detecting or quantifying the expressed polypeptide.The mutant ESR1 polypeptide can be detected and quantified by any of anumber of means known to those of skill in the art. These can includeanalytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,or various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, immunohistochemistry (IHC)and the like. A skilled artisan can adapt known protein/antibodydetection methods.

Another agent for detecting a mutant ESR1 polypeptide is an antibodymolecule capable of binding to a polypeptide corresponding to a markerof the invention, e.g., an antibody with a detectable label. Techniquesfor generating antibodies are described herein. The term “labeled,” withregard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin.

In another embodiment, the antibody is labeled, e.g., a radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. Inanother embodiment, an antibody derivative (e.g., an antibody conjugatedwith a substrate or with the protein or ligand of a protein-ligand pair(e.g., biotin-streptavidin)), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a mutant ESR1 protein, is used.

Mutant ESR1 polypeptides from cells can be isolated using techniquesthat are known to those of skill in the art. The protein isolationmethods employed can, for example, be such as those described in Harlowand Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Means of detecting proteins using electrophoretic techniques are wellknown to those of skill in the art (see generally, R. Scopes (1982)Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc., N.Y.).

In another embodiment, Western blot (immunoblot) analysis is used todetect and quantify the presence of a polypeptide in the sample.

In another embodiment, the polypeptide is detected using an immunoassay.As used herein, an immunoassay is an assay that utilizes an antibody tospecifically bind to the analyte. The immunoassay is thus characterizedby detection of specific binding of a polypeptide to an anti-antibody asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte.

The mutant ESR1 polypeptide is detected and/or quantified using any of anumber of immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

Kits

In one aspect, the invention features, a kit, e.g., containing anoligonucleotide having a mutation described herein, e.g., anoligonucleotide that hybridizes specifically to a mutation in the ligandbinding domain of ESR1. Optionally, the kit can also contain anoligonucleotide that is the wildtype counterpart of the mutantoligonucleotide.

A kit featured in the invention can include a carrier, e.g., a meansbeing compartmentalized to receive in close confinement one or morecontainer means. In one embodiment the container contains anoligonucleotide, e.g., a primer or probe as described above. Thecomponents of the kit are useful, for example, to diagnose identify amutation in a tumor sample in a patient, and to accordingly identify anappropriate therapeutic agent to treat the cancer. The probe or primerof the kit can be used in any sequencing or nucleotide detection assayknown in the art, e.g., a sequencing assay, e.g., an NGS method, RT-PCR,or in situ hybridization.

A kit featured in the invention can include, e.g., assay positive andnegative controls, nucleotides, enzymes (e.g., RNA or DNA polymerase orligase), solvents or buffers, a stabilizer, a preservative, a secondaryantibody, e.g., an anti-HRP antibody (IgG) and a detection reagent.

An oligonucleotide can be provided in any form, e.g., liquid, dried,semi-dried, or lyophilized, or in a form for storage in a frozencondition.

Typically, an oligonucleotide, and other components in a kit areprovided in a form that is sterile. When an oligonucleotide, e.g., anoligonucleotide that contains a mutation in ESR1, e.g., a mutation inthe ligand binding domain of ESR1 as described herein, or anoligonucleotide complementary to an ESR1 mutation, is provided in aliquid solution, the liquid solution generally is an aqueous solution,e.g., a sterile aqueous solution. When the oligonucleotide is providedas a dried form, reconstitution generally is accomplished by theaddition of a suitable solvent. The solvent, e.g., sterile buffer, canoptionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining an oligonucleotide in a concentration suitable for use in theassay or with instructions for dilution for use in the assay. In someembodiments, the kit contains separate containers, dividers orcompartments for the oligonucleotide and assay components, and theinformational material. For example, the oligonucleotides can becontained in a bottle or vial, and the informational material can becontained in a plastic sleeve or packet. In other embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, an oligonucleotide composition is contained in abottle or vial that has attached thereto the informational material inthe form of a label. In some embodiments, the kit includes a plurality(e.g., a pack) of individual containers, each containing one or moreunit forms (e.g., for use with one assay) of an oligonucleotide. Forexample, the kit includes a plurality of ampoules, foil packets, orblister packs, each containing a single unit of oligonucleotide for usein a sequencing or detecting a mutation in a tumor sample. Thecontainers of the kits can be air tight and/or waterproof. The containercan be labeled for use.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds specifically to a mutantESR1 polypeptide; and, optionally, (2) a second, different antibodywhich binds to either the polypeptide or the first antibody and isconjugated to a detectable agent.

In one embodiment, the kit can include informational material forperforming and interpreting the sequencing or diagnostic. In anotherembodiment, the kit can provide guidance as to where to report theresults of the assay, e.g., to a treatment center or healthcareprovider. The kit can include forms for reporting the results of asequencing or diagnostic assay described herein, and address and contactinformation regarding where to send such forms or other relatedinformation; or a URL (Uniform Resource Locator) address for reportingthe results in an online database or an online application (e.g., anapp). In another embodiment, the informational material can includeguidance regarding whether a patient should receive treatment with aparticular chemotherapeutic drug, depending on the results of the assay.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawings, and/or photographs,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about the sequencingor diagnostic assay and/or its use in the methods described herein. Theinformational material can also be provided in any combination offormats.

In some embodiments, a biological sample is provided to an assayprovider, e.g., a service provider (such as a third party facility) or ahealthcare provider, who evaluates the sample in an assay and provides aread out. For example, in one embodiment, an assay provider receives abiological sample from a subject, such as a blood or tissue sample,e.g., a biopsy sample, and evaluates the sample using an assay describedherein, e.g., a sequencing assay or in situ hybridization assay, anddetermines that the sample contains a nucleic acid containing a mutationdescribed in the Example. The assay provider, e.g., a service provideror healthcare provider, can then conclude that the subject is, or isnot, a candidate for a particular drug or a particular cancer treatmentregimen.

The assay provider can provide the results of the evaluation, andoptionally, conclusions regarding one or more of diagnosis, prognosis,or appropriate therapy options to, for example, a healthcare provider,or patient, or an insurance company, in any suitable format, such as bymail or electronically, or through an online database. The informationcollected and provided by the assay provider can be stored in adatabase.

The invention is further illustrated by the following example, whichshould not be construed as further limiting.

EXAMPLES Example 1: Massively Parallel Sequencing Assays to IdentifyNovel Alterations

The following exemplifies the use of massively parallel sequencingassays to identify novel alterations, such as mutations in the estrogenreceptor gene ESR1.

Massively parallel sequencing technology was used to examine eightformalin fixed paraffin embedded (FFPE) samples of breast cancermetastases in bone, lung and liver (Table 1). This assay can identifyall classes of DNA alterations (e.g., base substitutions, insertions anddeletions, copy number alterations and rearrangements) in a singlediagnostic test.

The sequencing analysis identified a 6-nucleotide non-frameshiftdeletion in the ESR1 gene in the ligand-binding domain (see FIG. 1). Themutation is a deletion of nucleotides 1046-1051 according to SEQ IDNO:1, which results in the deletion of amino acids LAD at positions 349to 351 of SEQ ID NO:2 (FIG. 2B), and insertion of a histidine atposition 349 of SEQ ID NO:4 (FIG. 4B). To our knowledge, this region ofthe ligand binding domain of ESR1 has not previously been identified asa recurrent site of mutation in human breast cancers.

The novel 6-nucleotide deletion occurs in a region of the ligand bindingdomain previously associated with resistance to treatment with SERMs(Selective Estrogen Receptor Modulators), such as tamoxifen. Forexample, a D351Y or D351E mutation results in a receptor that exhibitsan estrogenic response to SERMs, including raloxifene, EM652, GW7604,keoxifene and tamoxifen (Herynk and Fuqua, Endocrine Reviews 25:869-898,2004). The effects of the pure antiestrogen fulvestrant (Faslodex®) areunaffected by mutating this site to tyrosine. Experimental mutagenesisof this site to glycine, valine, or phenylalanine does not result in areceptor that responds to antiestrogens in an agonistic manner. Theantiestrogenic response of the D351Y mutant to antiestrogens requiresthe AF-1 domain because mutants with a deleted AF-1 domain lose theability to increase ER transactivation in response to the antiestrogenstamoxifen and raloxifene. Although the AF-2 domain is not required,D351Y mutants possessing intact AF-1 and AF-2 domains produce asynergistic response to antiestrogens. Further, the D351Y mutant showsdecreased interactions with the corepressors NCoR and SMRT (Herynk andFuqua, Endocrine Reviews 25:869-898, 2004).

In vitro assays and crystallographic studies indicated that D351 iscritical for the interaction with the antiestrogenic side chains ofSERMs. Liu et al. (J. Biol. Chem., 277:9189-9198, 2002) demonstratedthat the raloxifene side chain both shields and neutralizes the negativecharge at D351.

The location of the 6-nucleotide deletion suggests that this mutationmay also cause resistance to SERMs, such as tamoxifen and raloxifene.

Example 2: Methods

The following exemplifies certain embodiments of the methods andexperimental conditions used to identify the ESR1 mutation described inExample 1. Additional ESR1 screening can be done using, e.g., qRT-PCRanalysis of cDNA prepared from a pre-selected tumor sample.

Massively parallel DNA sequencing was done on hybridization captured,adaptor ligation-based libraries using DNA isolated from archived fixedparaffin-embedded tissue. A combination of analysis tools were used toanalyze the data and assign DNA alteration calls.

Genomic DNA Sequencing

Sequencing of cancer genes was done using DNA from archived formalinfixed paraffin embedded (FFPE) tumor specimens from breast cancerpatients. Sequencing libraries were constructed by the adapter ligationmethod using genomic DNA followed by hybridization selection withoptimized RNA hybridization capture probes (Agilent SureSelect customkit). Sequencing on the HiSeq2000 instrument (Illumina) was done using49×49 paired reads to an average depth of 514×. Data processing andmutation assignments for base substitutions, indels, copy numberalterations and genomic rearrangements were done using a combination oftools optimized for mutation calling from tumor tissue.

The sequencing results are summarized in the Tables below.

TABLE 1 Summary of Sequencing Results DNA Mean Large-scale SignificantTissue extraction exon genomic copy number ID yield coveragerearrangements gains/losses Novel Variants Bone 11 80 757 nonePTCH2:NM_001166292: c.1864C>A_p.H622N (0.40, 617), PTPRD:NM_130393:c.2095A>G_p.I699V (0.66, 589), ATM_c.378T>A_p.D126E (0.30, 759) Bone 12150 962 SPAG5-RARA ERBB2_gain DOT1L:NM_032482: rearrangement (2.4x)c.4210C>T_p.P1404S (SPAG5 (0.50, 1939), breakpoint in ERCC2:NM_000400:intron2, RARA c.1758+1C>G:splice breakpoint in (0.11, 551), exon 4),likely ESR1:NM_001122742: artifact of HER2 c.437C>A_p.P146Q amp (0.56,671), LTK:NM_206961: c.680C>T_p.P227L (0.48, 1559), PKHD1:NM_138694:c.10515C>A_p.S3505R (0.42, 866), PTCH1:NM_001083602: c.1610G>A_p.R537H(0.45, 958), PTPRD:NM_130393: c.91G>A_p.V31I (0.47, 754),RPTOR:NM_020761: c.3682G>A_p.V1228M (0.60, 1206), TSC1:NM_001162427:c.1607A>G_p.K536R (0.51, 703), ATM_c.5557G>A_p.D1853N (0.43, 607) Bone 830 53 n/a-low CD79A:NM_001783: coverage c.371G>A_p.R124H (0.20, 35),CDH20:NM_031891: c.1925T>C_p.M642T (0.40, 50), FGFR3:NM_022965:c.1936G>A_p.D646N (0.46, 67), IRS2:NM_003749: c.3098c>G_p.P1033R (0.14,104), JAK2:NM_004972: c.2538G>C_p.E846D (0.56, 62), KDR:NM_002253:c.1066G>A_p.G356S (0.15, 48), MDM2:NM_002392: c.400C>T_p.H134Y (0.20,65), MLL:NM_005933: c.2434A>G_p.K812E (0.13, 54), MUTYH:NM_001048171:c.1234C>T_p.R412C (0.42, 31), PAX5:NM_016734: c.39_39delG:frameshift(0.10, 170), PDGFRB:NM_002609: c.1504C>T_p.R502W (0.22, 63),PDGFRB:NM_002609: c.1505G>A_p.R502Q (0.73, 63), PTCH1:NM_001083603:c.131a>G_p.E44G (0.09, 111), ATM_c.5557G>A_p.D1853N (1.00, 55) Bone 1130 813 CCND1_gain NF1:NM_001042492: (7.0x) c.2666C>G_p.T889R (0.53,1063), ATM_c.2119T>C_p.S707P (0.25, 378), ATM_c.5557G>A_p.D1853N (0.72,401), KIT_c.1621A>C_p.M541L (0.48, 683) Bone 5 100 814 noneCDK4:NM_000075: c.122A>G_p.N41S (0.51, 881), ERBB2:NM_001005862:c.3285T>A_p.D1095E (0.44, 663), NOTCH1:NM_017617: c.3836G>A_p.R1279H(0.48, 1461), PIK3CA:NM_006218: c.716T>G_p.L239R (0.07, 1008),SMO:NM_005631: c.47_48insGCT:nonframeshift (0.18, 1004),TSC2:NM_001114382: c.275A>T_p.E92V (0.47, 841), ATM_c.4138C>T_p.H1380Y(0.50, 867), ATM_c.5557G>A_p.D1853N (0.47, 618) Bone 6 35 263 CTNNB1FGFR1_gain(2.3x) BRCA2:NM_000059: truncation PAX5_loss(0.5x)c.4436G>C_p.S1479T (0.64, 346), CDKN2A:NM_001195132: c.254C>A_p.A85D(0.83, 475), DOT1L:NM_032482: c.4327G>T_p.G1443C (0.55, 487),ESR1:NM_001122742: c.1046_1051delTGGCAG: nonframeshift (0.20, 211),ESR1:NM_001122742: c.656A>G_p.Y219C (0.62, 184), LRP1B:NM_018557:c.1105A>G_p.T369A (0.11, 143), LRP1B:NM_018557: c.3259G>A_p.G1087S(0.10, 146), NOTCH1:NM_017617: c.3836G>A_p.R1279H (1.00, 601),NTRK1:NM_002529: c.482G>A_p.R161H (0.49, 147), PTCH2:NM_001166292:c.3269C>T_p.A1090V (0.47, 221), ATM_c.5557G>A_p.D1853N (0.99, 173),MSH2_c.965G>A_p.G322D (0.52, 327) Liver 10 40 178 FGFR1_gain(2.2x),BCL6:NM_001130845: AKT3_gain(2.1) c.492G>T_p.E164D (0.49, 173),CHEK2:NM_007194: c.480A>G_p.I160M (0.63, 154), FANCA:NM_000135:c.1874G>C_p.C625S (0.63, 123), FGFR1:NM_001174065: c.266A>G_p.Q89R(0.16, 460), FLT4:NM_002020: c.1936G>A_p.E646K (0.50, 145),MCL1:NM_182763: c.134G>A_p.R45Q (0.44, 248), PIK3R1:NM_181504: c.555_560delGTTTCA: nonframeshift(0.25, 362), JAK3_c.2164G>A_p.V722I (0.43, 84)Lung 1 30 275 ERBB2_gain(3.1x), BAP1:NM_004656: CCND1_gain(2.9x)c.1924G>A_p.E642K (0.11, 273), DDR2:NM_006182: c.1323G>A_p.M441I (0.44,255), ERBB4:NM_005235: c.1122T>G_p.H374Q (0.47, 221), IKBKE:NM_014002:c.1912G>A_p.V6381 (0.57, 268), INHBA:NM_002192: c.41G>T_p.C14F (0.50,237), LRP1B:NM_018557: c.7420G>A_p.G2474S (0.51, 249), PIK3CG:NM_002649:c.400C>G_p.Q134E (0.53, 314), PTPRD:NM_130393: c.155G>T_p.R52I (0.15,294), PTPRD:NM_130393: c.3073A>G_p.R1025G (0.06, 217), SMO:NM_005631:c.173C>T_p.P58L (0.45, 355), TET2:NM_001127208: c.100C>T_p.L34F (0.46,205), USP9X:NM_001039591: c.3322G>A_p.D1108N (0.19, 366),ATM_c.5557G>A_p.D1853N (0.55, 213)

TABLE 2 Somatic Mutations Identified Tissue ID Somatic Mutations Bone 11TP53_c.581T>G_p.L194R(0.27, 480) Bone 12 Bone 8PIK3CA_c.1633G>A_p.E545K(0.44, 87), TP53_c.991C>T_p.Q331*(0.30, 37),CDH1:NM_004360: c.1531C>T_p.Q511*(0.29, 31) Bone 1PIK3CA_c.3140A>T_p.H1047L(0.30, 902),CDH1:NM_004360:c.859_866del8:frameshift(0.40, 404) Bone 5PIK3CA_c.3132T>A_p.N1044K(0.07, 931) Bone 6 TP53_c.489C>A_p.Y163*(0.80,342) Liver 10 CDH1:NM_004360:c.841_859del19:frameshift(0.14, 152),TP53:NM_001126114:c.634_641del8:frameshift(0.24, 110) Lung 1LRP1B:NM_018557:c.11818C>T_p.Q3940*(0.08, 346),TP53:NM_001126114:c.443_444insA:frameshift(0.14, 260)

Example 3: Mutations Identified in ESR1 Hinge Region and Ligand BindingDomain

Several mutations in the ligand binding domain of ESR1 were identifiedin large-scale sequencing studies performed using the protocolsdescribed herein. These mutations were identified in various tumorsamples, including breast, colorectal and non-small cell lung cancer,and are summarized in Table 3.

TABLE 3 Mutations identified in ESR1 ligand-binding domain. Tumor AminoAcid Nucleotide Other mutations identified Study Type Mutation^(a)Mutation^(b) Cancer Characteristics^(c) in sample^(c) A Colorectal T311MC932T ? ? S341L C1022T ? ? A350E C1049A A Colorectal R394H G1181A BBreast Q414* C1240T C NSCLC S433P T1297C D Breast R503W C1507T Primarybreast cancer, Her2 amp, P53 ER⁻ D Breast Y537N T1609A Metastatic, ER⁺,P53, BRCA, NF1, EGFR/MYC patient received gains tamoxifen E Breast Y537CA1610G Metastatic, ER⁺, PIK3CA, CCND1 treatment unknown D Breast Y537CA1610G Metastatic, ER⁺, BRCA patient received tamoxifen E Breast D538GA1613G Metastatic, ER⁺, CDH1, matching primary treatment unknown tumorsample was ESR1 WT D Breast D538G A1613G Metastatic, ER⁺, BRCA patientreceived tamoxifen Breast C insertion GCT Metastatic, ER⁺ betweeninsertion G344_L345 between 1033_1034 ^(a)amino acid mutaions refer toamino acid positions as defined by SEQ ID NO: 2 (FIG. 2B).^(b)nucleotide mutations refer to nucleotide position as defined by SEQID NO: 1 (FIG. 2B). ^(c)ER⁻ = estrogen receptor negative; ER⁺ = estrogenreceptor positive, as determined by immunohistochemistry (IHC)); “amp” =amplification

In particular, Table 3 summarizes the following: the tumor type, theamino acid and nucleotide positions of the mutations, the cancercharacteristics, and other mutations identified in the sample. Theposition of the amino acid and the nucleotide mutations are abbreviatedby the following shorthand. For example, a T311M amino acid mutationrefers to a change at position 311 of the amino acid sequence of SEQ IDNO:2 (FIG. 2B) from wild-type T or threonine amino acid to a mutant M ormethionine amino acid. Similarly, a C932T nucleotide mutation refers toa change at position 932 of the nucleotide sequence of SEQ ID NO:2 (FIG.2B) from wild-type C nucleotide to a mutant T nucleotide.

Most of the mutations identified were localized in the ligand bindingdomain of ESR1, with the exception of Residue 311 of SEQ ID NO:2 (FIG.2B), which was located near the C-terminal end of the hinge region ofESR1 (as depicted in FIG. 1). Other mutations identified includedmutations at positions 341, 350, 394, 414, 433, 503, 537 and 538 of SEQID NO:2 (FIG. 2B), and an insertion between amino acids 344 and 345 ofSEQ ID NO:2 (FIG. 2B).

Notably, several recurrent mutations at amino acid positions 537 and 538of the ligand binding domain of ESR1 were identified in the breastcancer samples. More specifically, three mutations of the tyrosineresidue at position 537 were identified in the breast cancer samples,for example, Y537N and Y537C. As shown in Table 3, all three mutationsare associated with metastatic, ER+ (positive) breast cancer. In two ofthe three samples, the patients received tamoxifen. The drug treatmentin one of the patients is unknown. At least two breast samples had arecurrent mutation at position 538 from D to G (see Table 3). Based onthe information available, at least two of the samples were metastatic,ER+. In one of the samples, the patient received tamoxifen and inanother sample, the previous treatment is unknown. Each of thesemutations is described in more detail below.

3/12 (25% of metastatic, ER⁺ (estrogen receptor positive),tamoxifen-treated breast cancers in study D showed mutations at aminoacids 537 and 538. Notably, 0/4 metastatic ER⁻, non-hormone-therapytreated breast cancers in study D showed these mutations; 0/33 primarybreast cancers (mixed ER/PR/HER2) in study D showed mutations; and 0/81primary triple negative breast cancers in study H showed mutations.

2/29 (7%) of metastatic, ER⁺, treatment unknown breast cancers in studyE showed mutations at amino acids 537 or 538. Notably again, 0/5metastatic, ER⁻ cases in study E had mutations at amino acids 537 or538. One ESR1 mutation was observed in a metastatic sample of a primarytumor/metastatic pair where the primary sample was ESR1 wild-type.

At least one D538G mutation occurred after treatment with tamoxifen. Inanother case, the D538G mutation was found in the metastatic tumor,while the primary tumor showed wild-type ESR1. In particular, sequencingof DNA from the primary tumor sample indicated that the ESR1 gene didnot carry the D538G mutation (see Study E result in Table 3 above).Thus, the presence of the D538G mutation can be associated with aresponse to SERM therapy, and/or be associated with a metastatictransition.

Further analysis of patient tumor (e.g., breast) tumor samples for thepresence of the ESR1 mutations disclosed herein is summarized in Table4. Generally, tumor samples carrying the ESR1 mutations disclosedherein, e.g., ESR1 mutations at amino acids 537 and/or 538, correlatedwith a late stage (e.g., metastatic) tumor. For example, out of 25patients with ER+ status, having metastatic cancer, 20% of patients hadsomatic mutations in ESR1 (e.g., ESR1 mutations at amino acids 537and/or 538), as shown in Table 4.

ESR1 somatic mutations are rare in breast cancer. The mutations at aminoacid 537 and 538 were observed primarily in ER+ samples. Tyrosine 537and Aspartate 538 are located at the C-terminal portion of the hormonebinding domain of ER, which are regions associated with dimerization andAF-2 function. Tyrosine 537 is a target of c-Src phosphorylation, andmutations at position 537, in particular, the Y537N substitution, havebeen associated with constitutive ER activity, as measured by activationof target gene expression in vitro (Barone et al. (2010) Clin CancerRes. 16(10):2702-8; Zhang et al. (1997) Cancer Res. 57:1244-1249).Activity of the 537 mutant ER was minimally affected by estradiol ortamoxifen, suggesting that phosphorylation of the tyrosine residue atposition 537 may regulate ER-α transactivation. Leucine at position 539of SEQ ID NO:2 is one of the direct amino acid contacts with estrogenligands, and mutations of L539 impair ER signaling (Shiau et al. (1998)Cell 95(7):927-937). Given the location, the mutation at position 538(e.g., the D538G mutation) can impair one or more of: thephosphorylation of tyrosine 537, e.g., by disrupting a kinase motif, ormay impact the binding of a co-activator or a ligand to the ER.

These results described herein suggest that subjects who are ER⁺ canbenefit from further testing to determine the presence or absence of amutation in the ER ligand binding domain, e.g., mutations at amino acid537 and/or 538. The presence of these mutations may indicate thepresence of constitutive activated ER receptor and/or the presence of ahormone-resistant cancer. Alternative therapies can be administered tosuch subjects, including an aromatase inhibitor (e.g., anastrozole), aSelective Estrogen Receptor Downregulator (SERD) or an anti-estrogen(e.g., fulvestrant), or an mTOR (mammalian Target of Rapamycin) pathwayinhibitor, or a combination thereof. Exemplary mTOR inhibitors include,for example, RAD001 (everolimus), CCI-799 (tensirolimus), and AP23573(ARIAD).

In other embodiments, the subjects can have mutations in genes otherthan the ER receptor that can be treated with therapeutic agentsspecific to the mutations. Examples of such mutations can be found inTable 3, and include a HER2 mutation (e.g., HER2 amplification), a p53mutation, BRCA, NF1, EGFR/myc gains, PIK3CA, CCND1 and CDH1. Thus, thesubjects can receive a therapy that target the ESR1 mutation, alone orin combination with a therapy that targets another mutation identifiedas part of the cancer, e.g., a drug that targets mutant HER2 mutation(e.g., HER2 amplification), a p53 mutation, BRCA, NF1, EGFR/myc gains,PIK3CA, CCND1 and/or CDH1.

TABLE 4 Further analysis of ESR1 alterations # of % of patients patientswith known with known somatic somatic mutations mutations Specimens inESR1 or in ESR1 or # patients ER status sequenced amp amp ESR1alteration 17 + Primary 1 6% ESR1 alteration is AMP, estimated CN = 1025 + Metastasis 5 20%  all ESR1 mutations aa537/538 16 − Primary 0 0% 5− Metastasis 0 0% 1 uncertain Metastasis 0 0% 33 + Primary/Met. 2 6%ESR1 aa537 matched and 538 present set in metastases, absent in primary1 + Primary 0 0% 6 − Primary/Met. 0 0% matched set 3 − Primary 0 0% 16 +Primary/Met. 1 6% ESR1 amp matched present in all 3 set metastaticsamples in set, ESR1 Y537N present in 2/3, no matching primary sequenced3 + Primary 0 0% 118 unknown in mixed 6 5% 5 ESR1 D538G, general 1 AMP;3/5 with D538G known as mets of ER+ Breast Ca.; matching primarysequenced for 2/5 and confirmed negative 81 − Primary 0 0% (TNBC)

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby the COSMIC database, available on the worldwide web atsanger.ac.uk/genetics/CGP/cosmia; and the Institute for Genomic Research(TIGR) on the world wide web at tigr.org and/or the National Center forBiotechnology Information (NCBI) on the world wide web atncbi.nlm.nih.gov.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

We claim:
 1. A method of treating a subject having a breast cancer, themethod comprising: acquiring knowledge of a presence of (a) a mutantestrogen receptor 1 (ESR1) polypeptide or a nucleic acid moleculeencoding the mutant ESR1 polypeptide and (b) an Estrogen Receptorpositive (ER+) breast cancer in said subject, wherein the mutant ESR1polypeptide or the nucleic acid molecule encoding the mutant ESR1polypeptide comprises a mutation chosen from one or more of: (i) one ormore of mutations at amino acid position 311, 341, 350, 394, 414, 433,or 503, or 538 of SEQ ID NO: 2; (ii) a deletion of nucleotides 1046-1051of SEQ ID NO: 1; (iii) an insertion of a histidine as shown at aminoacid position 349 of SEQ ID NO:4; (iv) an insertion between amino acidpositions 344 and 345 of SEQ ID NO: 2; or (v) a tyrosine to cysteinesubstitution at amino acid position 537 (a Y537C) of SEQ ID NO: 2; andresponsive to said knowledge, administering to the subject an effectiveamount of an estrogen receptor modulator set forth in Table 5, therebytreating the breast cancer in the subject.
 2. The method of claim 1,wherein the mutant ESR1 polypeptide comprises a mutation chosen from oneor more of: an aspartate to glycine substitution at amino acid position538 (a D538G) of SEQ ID NO: 2; a deletion of amino acids Leu-Ala-Asp(LAD) at positions 349-351 of SEQ ID NO: 2; a threonine to methioninesubstitution at amino acid position 311 (a T311M) of SEQ ID NO: 2; aserine to leucine substitution at amino acid position 341 (a S341L) ofSEQ ID NO: 2; an alanine to glutamate substitution at amino acidposition 350 (a A350E) of SEQ ID NO: 2; an arginine to histidinesubstitution at amino acid position 394 (a R394H) of SEQ ID NO: 2; aglutamine substitution at amino acid position 414 of SEQ ID NO: 2; aserine to proline substitution at amino acid position 433 (a S433P) ofSEQ ID NO: 2; an arginine to tryptophan substitution at amino acidposition 503 (a R503W) of SEQ ID NO: 2; or an insertion of a cysteinebetween amino acid positions 344 and 345 of SEQ ID NO:2.
 3. The methodof claim 1, wherein said subject has one or more of the following: (i)previously received a treatment with a SERM; (ii) has failed a first orsecond line of treatment with a SERM; (iii) has a late stage, metastaticprogressive breast cancer; (iv) is post-menopausal or pre-menopausal;(v) stops a treatment with a SERM and begins a treatment with ananti-cancer agent that is not a SERM; or (vi) is premenopausal andreceives an oophorectomy.
 4. The method of claim 3, wherein the SERM ischosen from raloxifene, EM652, GW7604, keoxifene, toremifene, tamoxifen,lasofoxifene, levormeloxifene, bazedoxifene, or arzoxifene.
 5. Themethod of claim 1, further comprising administering to the subject ananti-cancer agent chosen from a Selective Estrogen Receptor Degrader(SERD), an aromatase inhibitor, an mTOR pathway inhibitor, or achemotherapeutic agent.
 6. The method of claim 1, further comprisingadministering to the subject fulvestrant.
 7. The method of claim 5,wherein the aromatase inhibitor is chosen from aminoglutethimide,testolactone, anastrozole, letrozole, exemestane, vorozole, formestane,fadrozole, 4-hydroxyandrostenedione, 1,4,6-androstatrien-3,17-dione(ATD), or 4-Androstene-3,6,17-trione (“6-OXO”).
 8. The method of claim5, wherein the mTOR pathway inhibitor is chosen from rapamycin,temsirolimus, everolimus, ridaforolimus, AP23573, AZD8055, BEZ235,BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615,KU-0063794, WYE-354, INK128, temsirolimus (CCI-779), Palomid 529 (P529),PF-04691502, or PKI-587.
 9. The method of claim 1 wherein the estrogenreceptor modulator is administered in combination with a differenttherapeutic agent or a different therapeutic modality.
 10. The method ofclaim 9, wherein the different therapeutic agent or differenttherapeutic modality is selected according to a determination of apresence of a mutation other than the mutation in the mutant ESR1polypeptide or in the nucleic acid molecule encoding the mutant ESR1polypeptide, and wherein the mutation other than the mutation in themutant ESR1 polypeptide or in the nucleic acid molecule encoding themutant ESR1 polypeptide is chosen from a HER2 mutation, a p53 mutation,a BRCA mutation, an NF1 mutation, an EGFR/myc gain, a PIK3CA mutation, aCCND1 mutation, a CDH1 mutation, or a combination thereof.
 11. Themethod of claim 1, wherein the acquiring knowledge step comprisesdetermining the presence of the mutation in the mutant ESR1 polypeptideor in the nucleic acid molecule encoding the mutant ESR1 polypeptide bysequencing.
 12. The method of claim 1, wherein the subject waspreviously tested for the presence of a mutant ESR1 polypeptide or anucleic acid molecule encoding the mutant ESR1 polypeptide, wherein themutant ESR1 polypeptide or the nucleic acid molecule encoding the mutantESR1 polypeptide was not detected during the previous testing, andwherein the subject was administered a SERM based on the knowledge thatthe mutant ESR1 polypeptide or the nucleic acid molecule encoding themutant ESR1 polypeptide was not detected during the previous testing.13. The method of claim 1, wherein the subject was previously tested atintervals for the presence of a mutant ESR1 polypeptide or a nucleicacid molecule encoding the mutant ESR1 polypeptide, and wherein themutant ESR1 polypeptide or the nucleic acid molecule encoding the mutantESR1 polypeptide was not detected during the previous testing, andwherein the subject continued a treatment with a SERM based on theknowledge that the mutant ESR1 polypeptide or the nucleic acid moleculeencoding the mutant ESR1 polypeptide was not detected during theprevious testing.
 14. The method of claim 1, wherein the subject waspreviously tested or is currently being tested for the presence of themutant ESR1 polypeptide or the nucleic acid molecule encoding the mutantESR1 polypeptide at six-month or one-year intervals.
 15. The method ofclaim 1, wherein the subject was tested at intervals for the presence ofa mutant ESR1 polypeptide or a nucleic acid molecule encoding the mutantESR1 polypeptide, and wherein the mutant ESR1 polypeptide or the nucleicacid molecule encoding the mutant ESR1 polypeptide was detected duringthe testing, and wherein the subject stopped a treatment with a SERMbased on the knowledge that the mutant ESR1 polypeptide or the nucleicacid molecule encoding the mutant ESR1 polypeptide was detected duringthe testing.
 16. The method of claim 1, wherein the acquiring knowledgestep comprises detecting the mutant ESR1 polypeptide or the nucleic acidmolecule encoding the mutant ESR1 polypeptide in a sample from thesubject.
 17. The method of claim 1, further comprising: identifying,selecting, or obtaining information or knowledge that the subject hasparticipated in a clinical trial or has been treated for cancer;acquiring genotype information that identifies the mutant ESR1polypeptide or the nucleic acid molecule encoding the mutant ESR1polypeptide as being in the subject, wherein the presence of the mutantESR1 polypeptide or the nucleic acid molecule encoding the mutant ESR1polypeptide identifies the subject as having an increased risk for, orhaving, a cancer associated with the mutant ESR1 polypeptide or thenucleic acid molecule encoding the mutant ESR1 polypeptide.
 18. Themethod of claim 1, wherein the mutant ESR1 polypeptide comprises adeletion of amino acids Leu-Ala-Asp (LAD) at positions 349-351 of SEQ IDNO:
 2. 19. The method of claim 1, wherein the nucleic acid moleculeencoding the mutant ESR1 polypeptide comprises a deletion of nucleotides1046-1051 of SEQ ID NO:
 1. 20. The method of claim 5, wherein theanti-cancer agent is an mTOR pathway inhibitor.
 21. The method of claim1, wherein the estrogen receptor modulator set forth in Table 5 is(E)-3-(4-((E)-2-(2-Chloro-4-fluorophenyl)-1-(1H-indazol-5-yl)but-1-en-1-yl)phenyl)acrylicacid.
 22. The method of claim 16, wherein the nucleic acid moleculeencoding the mutant ESR1 polypeptide comprises a deletion of nucleotides1046-1051 of SEQ ID NO:
 1. 23. The method of claim 16, wherein thesample comprises a tissue, a tumor biopsy, blood, serum, plasma, urine,circulating tumor cells, circulating nucleic acids, or bone marrow. 24.The method of claim 16, wherein the sample is a nucleic acid or aprotein sample.
 25. The method of claim 16, comprising contacting aprotein sample with a reagent which specifically binds to the mutantESR1 polypeptide; and detecting the formation of a complex of the mutantESR1 polypeptide and the reagent.
 26. The method of claim 16, whereinthe nucleic acid molecule encoding the mutant ESR1 polypeptide isdetected by a method chosen from one or more of: a nucleic acidhybridization assay, polymerase chain reaction (PCR), PCR-RFLP assay,real-time PCR, sequencing, metaphase cytogenetic analysis, FISH, breakaway FISH, spectral karyotyping, MFISH, comparative genomichybridization, in situ hybridization, SSP, or mass-spectrometricgenotyping.